Inhibitors of calcium-activated chloride channels

ABSTRACT

Provided herein are methods for identifying compounds that are inhibitors of a calcium-activated chloride channel. Aminothiophene and aminothiazole compounds, and compositions comprising these compounds, described herein that inhibit efflux of chloride through a calcium-activated chloride channel are useful for treating diseases, disorders, and sequelae of diseases, disorders, and conditions that are associated with aberrantly increased chloride and fluid secretion, for example, secretory diarrhea.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation patent application of U.S.application Ser. No. 12/747,468, having a filing date of Sep. 15, 2010;which is a national stage application filed under 35 U.S.C. §371 ofInternational Patent Application No. PCT/US2008/086600 accorded aninternational filing date of Dec. 12, 2008; which claims the benefitunder 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/013,988,filed Dec. 14, 2007, all of which applications are incorporated hereinby reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant numbersDK72517, HL73854, EB00415, EY13574, DK35124, and DK43840 from theNational Institutes of Health. The government has certain rights in thisinvention.

BACKGROUND

1. Field

Agents are needed for treating diseases and disorders related toaberrant function of calcium-activated chloride channels, including, forexample, increased intestinal fluid secretion, secretory diarrhea,asthma, and cystic fibrosis. Small molecule compounds that are potentinhibitors of chloride conductance via calcium-activated chloridechannels and methods for identifying such compounds are describedherein.

2. Description of the Related Art

Diarrheal disease in children is a global health concern: approximatelyfour billion cases among children occur annually, resulting in twomillion deaths. Antibiotics are routinely used to treat diarrhea;however, the antibiotics are ineffective for treating many pathogens,and the use of these drugs contributes to development of antibioticresistance in other pathogens.

Calcium-activated chloride channels (CaCCs) are believed to provide animportant route for chloride (Cl⁻¹) and fluid secretion in secretorydiarrheas. Excess transepithelial salt and water transport by one ormore CaCCs may be caused by certain drugs (e.g., antiretrovirals,chemotherapeutics) and viruses (see, e.g., Lorrot et al., Virol. J. 4:31(2007); Morris et al., Am. J. Physiol. 277:G431-44 (1999); Rufo et al.,Am. J. Physiol. 264:C998-1008 (2004); Schultheiss et al., Eur. J.Pharmacol. 546:161-70 (2006); Takahashi et al., J. Med. Microbiol.49:801-10 (2000); Schultheiss et al., J. Membr. Biol. 204:117-27 (2005);Gyömörey et al., Pflugers Arch. 443 Suppl 1:S103-6 (2001); Kidd et al.,Annu. Rev. Physiol. 62:493-513 (2000); Barrett et al., Annu. Rev.Physiol. 62:535-72 (2000)). The morbidity and mortality associated withsecretory diarrhea indicate an imperative need for potent inhibitors ofCaCCs.

In addition, CaCCs are believed to provide an important route forchloride (Cl⁻) and fluid secretion in pulmonary diseases and disorders.For example, smooth muscle CaCCs have been implicated in thepathophysiology of asthma (see, e.g., Bolton et al., J. Physiol.570:5-11 (2006); Farthing, Dig. Dis. 24:47-58 (2006); Thiagarajah etal., Trends Pharmacol. Sci. 26, 172-75 (2005))). Airway CaCCs have beenidentified in some model systems to be upregulated in cystic fibrosis(Tarran et al., J. Gen. Physiol. 120:407-18 (2002)), providingalternative chloride conductance to compensate for missing or defectiveCFTR. However, CaCC activity may contribute to excess mucus secretion byepithelial cells in the lungs of patients with pulmonary diseases anddisorders, such as asthma, chronic obstructive pulmonary disease (COPD),bronchiectasis, and cystic fibrosis. Excess mucus production andconcomitant change in the lung environment lead to colonization bybacteria that exacerbate the pathophysiology of the diseased lung. Thus,inhibitors of CaCCs are needed treating pulmonary diseases and disordersthat exhibit mucus hypersecretion.

BRIEF SUMMARY

Briefly stated, provided herein are compounds, compositions, and methodsthat are useful for treating diseases and disorders related to orassociated with aberrantly increased CaCC chloride secretion from cells.Also provided herein are methods for identifying and characterizingagents, including compounds, that inhibit calcium-activated chloridechannels.

In one embodiment, compounds, and compositions comprising thesecompounds, of the aminothiophene class are provided. In one embodiment,the composition comprises a physiologically acceptable excipient and acompound having the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein R¹ is hydrogen or optionally substituted alkyl; R² is hydroxy,optionally substituted alkoxy, or optionally substituted phenylamino; R³is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted cycloalkyl, optionally substituted phenyl, oroptionally substituted heterocyclyl; and n is 0, 1, or 2, and whereinthe compound of structure I comprises at least one —COOH. Also providedherein in certain embodiments (described in greater detail herein), areaminothiophene compounds and compositions comprising these compoundswherein the aminothiophene compounds have a substructure of formulae(IA) and (Ia)-(Ii).

In another embodiment, compounds and compositions comprising thesecompounds, of the aminothiazole class are provided. In one embodiment,the composition comprises a physiologically acceptable excipient and acompound having the following structure (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein R⁷ is optionally substituted C₁₋₆ alkyl, optionally substitutedphenyl, or optionally substituted phenylacyl; R⁸ is hydrogen, optionallysubstituted C₁₋₆ alkyl, or optionally substituted phenyl; R⁹ and R¹⁰ arethe same or different and independently hydrogen, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substituted phenoxy.Also provided herein in certain embodiments (described in greater detailherein), are aminothiazole compounds and compositions comprising thesecompounds wherein the aminothiazole compounds have a substructure offormulae (IIa)-(IIh).

Also provided herein is a method of inhibiting a calcium-activatedchloride channel comprising: contacting (a) a cell that comprises thecalcium-activated chloride channel and (b) a compound or a compositioncomprising the compound wherein the compound is (i) an aminothiophenecompound of structure (I), including substructures (IA) and (Ia)-(Ii)described above and herein and/or (ii) an aminothiazole compound ofstructure (II), including substructures (IIa)-(IIh) as described aboveand herein, in an amount effective and under conditions and for a timesufficient to inhibit activation of the channel. In a specificembodiment, the cell is an epithelial cell. In a particular embodiment,the epithelial cell is an intestinal epithelial cell or a lungepithelial cell. In a specific embodiment, the calcium-activatedchloride channel is TMEM16A, and in other specific embodiments, theTMEM16A calcium-activated chloride channel is a human TMEM16Acalcium-activated chloride channel.

In one embodiment, provided herein is a method of inhibiting fluidsecretion from a cell comprising administering to a subject of thecomposition comprising physiologically acceptable excipient and (i) anaminothiophene compound of structure (I), including substructures (IA)and (Ia)-(Ii) described above and herein and/or (ii) an aminothiazolecompound of structure (II), including substructures (IIa)-(IIh), in anamount effective to inhibit conductance of chloride through acalcium-activated chloride channel, thereby inhibiting fluid secretionfrom the cell, wherein the subject has a condition, disease or disorderthat is treatable by inhibiting conductance of chloride through acalcium-activated chloride channel. In certain embodiments, the diseaseor disorder is selected from abnormally increased intestinal fluidsecretion, secretory diarrhea, asthma, chronic obstructive pulmonarydisease, bronchiectasis, or cystic fibrosis. In other embodiments, acondition that is treatable by inhibiting conductance of chloridethrough a calcium-activated chloride channel includes abnormallyincreased mucus secretion, which in certain embodiments is a conditionof a disease or disorder that is a pulmonary disorder (e.g., asthma,chronic obstructive pulmonary disease, bronchiectasis, or cysticfibrosis).

In another embodiment, a method of treating a condition, disease, ordisorder associated with abnormally increased chloride ion secretion isprovided, wherein the method comprises administering to a subject acomposition comprising a physiologically acceptable excipient and (i) anaminothiophene compound of structure (I), including substructures (IA)and (Ia)-(Ii) described above and herein and/or (ii) an aminothiazolecompound of structure (II), including substructures (IIa)-(IIh)described above and herein, in an amount effective to inhibit acalcium-activated chloride channel, thereby inhibiting chloride ionsecretion. In one certain embodiment, the disease or disorder isabnormally (i.e., aberrantly) increased intestinal fluid secretion. In aparticular embodiment, the disease or disorder is secretory diarrhea. Inanother particular embodiment, the condition, which may be a conditionof the disease or disorder described herein is abnormally increasedmucus secretion. In certain embodiments, a disease or disorder thatcomprises the condition of abnormally increased mucus secretion isasthma, chronic obstructive pulmonary disease, bronchiectasis, or cysticfibrosis. In certain embodiments, the method of treating a disease ordisorder further comprising administering to the subject an agent thatinhibits ion transport by a cystic fibrosis transmembrane conductanceregulator (CFTR). In a specific embodiment, the agent is athiazolidinone compound. In a more specific embodiment, thethiazolidinone compound is3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyemethylene]-2-thioxo-4-thiazolidinone.

Also provided herein is a use of a composition comprising (i) anaminothiophene compound of structure (I), including substructures (IA),(Ia)-(Ii) described above and herein and/or (ii) an aminothiazolecompound of structure (II), including substructures (IIa)-(IIh)described above and herein, for treating a condition, disease, ordisorder associated with abnormally increased chloride ion secretionfrom a cell. In specific embodiments, the disease or disorder issecretory diarrhea, asthma, chronic obstructive pulmonary disease,bronchiectasis, or cystic fibrosis. In other certain embodiments, a useis provided that is a use of a composition comprising a physiologicallyacceptable excipient (i) an aminothiophene compound of structure (I),including substructures (IA), (Ia)-(Ii) described above and hereinand/or (ii) an aminothiazole compound of structure (II), includingsubstructures (IIa)-(IIh) described above and herein, and a compositioncomprising an agent that inhibits ion transport by a cystic fibrosistransmembrane conductance regulator (CFTR). In a specific embodiment,the agent is a thiazolidinone compound. In a more specific embodiment,the thiazolidinone compound is3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone.

In another embodiment, use of a composition is provided wherein the usecomprises (i) an aminothiophene compound of structure (I), includingsubstructures (IA), (Ia)-(Ii) described above and herein and/or (ii) anaminothiazole compound of structure (II), including substructures(IIa)-(IIh) described above and herein, for the manufacture of amedicament for treating a condition, disease, or disorder associatedwith abnormally increased chloride ion secretion from a cell. In acertain embodiment, the cell is an epithelial cell. In a particularembodiment, the epithelial cell is an intestinal or lung epithelialcell. In specific embodiments, the disease or disorder is secretorydiarrhea, asthma, chronic obstructive pulmonary disease, bronchiectasis,or cystic fibrosis. In other certain embodiments, a use is provided thatis a use of a composition comprising (i) an aminothiophene compound ofstructure (I), including substructures (IA), (Ia)-(Ii) described aboveand herein and/or (ii) an aminothiazole compound of structure (II),including substructures (IIa)-(IIh) described above and herein, and acomposition comprising an agent that inhibits ion transport by a cysticfibrosis transmembrane conductance regulator (CFTR) for the manufactureof the medicament. In a specific embodiment, the agent is athiazolidinone compound. In a more specific embodiment, thethiazolidinone compound is3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone.

Also provided herein is a method of identifying an agent that is aninhibitor of a calcium-activated chloride channel comprising: (a)contacting a cell and a candidate agent in a test sample, (underconditions and for a time sufficient) to permit interaction between thecandidate agent and the cell, wherein the cell comprises (i) acalcium-activated chloride channel and (ii) a cytoplasmic indicatorprotein that binds halide; (b) adding to the test sample (i) at leastone calcium-elevating agonist and (ii) iodide, under conditions and fora time sufficient for the calcium-elevating agonist to bind to the cell(i.e., to permit binding of the calcium-elevating agonist to the cell),wherein binding of the calcium-elevating agonist to the cell increasesthe level of calcium ion (Ca²⁺) in the cell; and (c) determining thelevel of iodide influx in the presence of the candidate agent andcomparing the level of iodide influx in the presence of the candidateagent with the level of iodide influx in the absence of the candidateagent, wherein a decrease in the level of iodide influx in the presenceof the candidate agent compared with the level of iodide influx in theabsence of the candidate agent, indicates that the candidate agent is aninhibitor of the calcium-activated chloride channel. In a certainembodiment, the cell is an epithelial cell. In a particular embodiment,the epithelial cell is an intestinal epithelial cell or a pulmonaryepithelial cell. In a more particular embodiment, the intestinalepithelial cell is an HT-29 cell. In certain embodiment, the steps ofthe method are performed in each of a plurality of reaction vessels in ahigh throughput screening array. In other particular embodiments, thecalcium-elevating agonist is selected from histamine, calcimycin, ATP,carbachol, and forskolin. In a more specific embodiment, the test sampleof step (b) is contacted with at least two, at least three, or at leastfour calcium-elevating agonists. In yet another specific embodiment, thetest sample of step (b) is contacted with at least two calcium-elevatingagonists, wherein the at least two calcium-elevating agonists are ATPand carbachol. In certain embodiments the calcium-activated chloridechannel is TMEM16A; in specific embodiments, the TMEM16A is a mammalianTMEM16A; in other certain embodiments the mammalian TMEM16A is a humanTMEM16A. In certain embodiments, the cytoplasmic indicator protein is ayellow fluorescent protein (YFP) variant (also called herein YFPmutant). In a particular embodiment, the YFP variant (also called hereinYFP mutant) is YFP-H148Q/I152L. In still another embodiment, theepithelial cell comprising the cytoplasmic indicator protein is obtainedby transforming, transfecting, or transducing the epithelial cell with arecombinant expression vector that comprises a polynucleotide thatencodes the cytoplasmic indicator protein. In one specific embodiment,the cell transiently expresses the cytoplasmic indicator protein. Inanother specific embodiment, the cell stably expresses the cytoplasmicindicator protein. In still other embodiments, the recombinantexpression vector is a plasmid or a viral vector. In a specificembodiment, the viral vector is a retroviral vector. In another specificembodiment, the retroviral vector is a lentiviral vector. In stillanother embodiment, step (c) comprises determining the level of iodideinflux in the presence and absence of the candidate agent at multipletime points.

In another embodiment, a method is provided for determining influx of ananion in a cell that comprises or is suspected of comprising acalcium-activated chloride channel, wherein the anion is halide or NO₃⁻, said method comprising: (a) contacting the cell with the anion in thepresence of a calcium-elevating agonist and in the absence of thecalcium-elevating agonist, under conditions and for a time sufficientthat permit interaction between the calcium-elevating agonist and theepithelial cell, wherein the cell comprises a cytoplasmic indicatorprotein that binds the anion, and wherein binding of thecalcium-elevating agonist to the cell increases the level of calcium ion(Ca²⁺) in the cell; and (b) determining the level of anion influx in thepresence of the calcium-elevating agonist and determining the level ofanion influx in the absence of the calcium-elevating agonist and thencomparing the level of anion influx in the presence of thecalcium-elevating agonist to the level of anion influx in the absence ofthe calcium-elevating agonist, thereby determining influx of the anionin the cell. In a specific embodiment, the cell comprising thecytoplasmic indicator protein is obtained by transforming, transfecting,or transducing the epithelial cell with a recombinant expression vectorthat comprises a polynucleotide that encodes the cytoplasmic indicatorprotein. In a certain embodiment, the cell is an epithelial cell. In aparticular embodiment, the epithelial cell is an intestinal epithelialcell or a pulmonary epithelial cell. In a more particular embodiment,the intestinal epithelial cell is an HT-29 cell. In certain embodimentsthe calcium-activated chloride channel is TMEM16A; in specificembodiments, the TMEM16A is a mammalian TMEM16A; in other certainembodiments the mammalian TMEM16A is a human TMEM16A. In one specificembodiment, the recombinant expression vector is a plasmid or viralvector. In another specific embodiment, the viral vector is a retroviralvector. In a certain embodiment, the retroviral vector is a lentiviralvector.

In another embodiment, a method is provided for determining influx of ananion in a cell that comprises or is suspected of comprising acalcium-activated chloride channel, wherein the anion is halide or NO₃⁻, said method comprising: (a) culturing the cell to provide a pluralityof cells; (b) transforming, transfecting, or transducing the pluralityof cells a expression vector that comprises a polynucleotide encoding anindicator protein that is capable of binding the anion; (c) culturingthe plurality of cells of step (b) under conditions and for a timesufficient that permit expression of the indicator protein in thecytoplasm of the cells; (d) contacting the plurality of cells with theanion in the presence and absence of a calcium-elevating agonist, underconditions and for a time sufficient to permit interaction between thecalcium-elevating agonist and the plurality of cells, wherein binding ofthe calcium-elevating agonist to the plurality of cells increases thelevel of calcium ion (Ca²⁺) in the plurality of cells; and (e)determining the level of anion influx in the presence of thecalcium-elevating agonist and determining the level of anion influx inthe absence of the calcium-elevating agonist, and then comparing thelevel of anion influx in the presence of the calcium-elevating agonistto the level of anion influx in the absence of the calcium-elevatingagonist, thereby determining influx of anion in the cell. In a certainembodiment, the cell is an epithelial cell. In a particular embodiment,the epithelial cell is an intestinal epithelial cell or a pulmonaryepithelial cell. In a more particular embodiment, the intestinalepithelial cell is an HT-29 cell. In certain embodiments thecalcium-activated chloride channel is TMEM16A; in specific embodiments,the TMEM16A is a mammalian TMEM16A; in other certain embodiments themammalian TMEM16A is a human TMEM16A. In one specific embodiment, therecombinant expression vector is a plasmid or viral vector. In anotherspecific embodiment, the viral vector is a retroviral vector. In acertain embodiment, the retroviral vector is a lentiviral vector. Inother specific embodiments, the indicator protein is a yellowfluorescent protein (YFP) mutant (also called herein YFP variant). Instill another specific embodiment, the YFP mutant is YFP-H148Q/I152L. Incertain embodiments, the step of determining the level of influx of theanion in the presence of the calcium-activated chloride channel agonistand determining the level of influx of the anion in the absence of thecalcium-activated chloride channel agonist are determined at multipletime points (i.e., the step of determining comprises determining thelevel of influx of the anion in the presence of the calcium-activatedchloride channel agonist and determining the level of influx of theanion in the absence of the calcium-activated chloride channel agonistat multiple time points over a time course). In still other embodiments,the calcium-elevating agonist is a first calcium-elevating agonist andis selected from histamine, calcimycin, ATP, carbachol, and forskolin.In a particular embodiment, the method further comprises a secondcalcium-elevating agonist. In certain particular embodiments, the firstcalcium-elevating agonist is ATP and the second calcium-elevatingagonist is carbachol. Also, in specific embodiments of the method, theanion is iodide.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an agent” includesa plurality of such agents, and reference to “a cell” or “the cell”includes reference to one or more cells and equivalents thereof (e.g.,plurality of cells) known to those skilled in the art, and so forth. Theterm “about” when referring to a number or a numerical range means thatthe number or numerical range referred to is an approximation withinexperimental variability (or within statistical experimental error), andthus the number or numerical range may vary between 1% and 15% of thestated number or numerical range. The term “comprising” (and relatedterms such as “comprise” or “comprises” or “having” or “including”) isnot intended to exclude in other certain embodiments, for example, thatan embodiment of any composition of matter, composition, method, orprocess, or the like, described herein, may “consist of” or “consistessentially of” the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. FIG. 1A illustrates a schematic of a cell-based,fluorescence high-throughput screening assay. In this example,calcium-activated chloride channel (CaCC)-facilitated iodide influx ismeasured from the kinetics of decreasing YFP-H148Q/I152L fluorescence inresponse to iodide addition to the extracellular solution. CaCC may beactivated by a mixture of calcium-elevating agonists, includingcarbachol (Cch); carbachol receptor (m₃AChR); purinergic receptor(P₂y₂); and calcium-calmodulin protein kinase 2 (CaMKII). FIG. 1B is afluorescence micrograph of lentivirus-infected HT-29 cells stablyexpressing the YFP iodide sensor.

FIG. 2A-2D present data illustrating time course experiments thatmeasure YFP fluorescence after extracellular iodide addition toYFP-expressing HT-29 cells. The scale bar on the y-axis indicates thepercentage reduction in fluorescence relative to baseline fluorescence(before iodide addition). As indicated in the figures, the solutionscontained histamine (100 μM), calcimycin (10 μM), ATP (100 μM),carbachol (100 μM), carbachol (100 μM)+CFTR_(inh)-172 (20 μM), orforskolin (20 μM), either individually as shown in FIG. 2A, or invarious combinations (at same concentrations) as shown in FIG. 2B. FIG.2C represents a carbachol concentration-response study. FIG. 2Ddemonstrates the initial negative fluorescence curve slope (followingextracellular iodide addition) as a function of time after carbachol/ATP(each 100 μM) addition and extracellular iodide addition.

FIG. 3A-3C. FIG. 3A (left) shows a time course of YFP fluorescence inHT-29 cells following iodide addition in the absence or presence ofcarbachol (100 μM) and ATP (100 μM). FIG. 3A (right) is a histogramdistribution of initial iodide influx rates (d[I⁻]/dt) determined frominitial fluorescence slopes. FIG. 3B (left) provides examples offluorescence data for individual compounds in the primary screen. FIG.3B (right) is a histogram distribution of percentage inhibition fromprimary compound screening. Dashed vertical line denotes selectioncriteria for further evaluation. FIG. 3C describes structures ofcompounds of classes A-F identified from the primary compound screen.

FIG. 4A-4C. FIG. 4A shows one example of the synthesis ofCaCC_(inh)-A01:6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid. FIG. 4B shows one example of the synthesis of CaCC_(inh)-B01:2-hydroxy-4-(4-p-tolylthiazol-2-ylaminobenzoic acid. FIG. 4C illustratesconcentration-inhibition data for CaCC_(inh)-A01 and CaCC_(inh)-B01determined by plate reader fluorescence assay.

FIG. 5A-5D. FIG. 5A is a time course of YFP fluorescence in HT-29 cellsfollowing iodide addition in the absence or presence of thapsigargin incells pre-treated with 30 μM of indicated compounds. FIG. 5B shows YFPfluorescence in FRT cells expressing human wildtype CFTR followingiodide addition in cells pre-treated with 30 μM of indicated compounds.CFTR_(inh)-172 (20 μM) was present where indicated. FIG. 5C showscalcium signaling measured by fura-2 fluorescence in response toindicated agonists and test compounds. A=CaCC_(inh)-A01;B=CaCC_(inh)-B01. Representative data shown on the left, with averageddata on the right (SE, n=3-4). FIG. 5D shows ATP/carbachol-inducedCaMKII phosphorylation determined by immunoblot analysis (representativeof 3 separate experiments).

FIG. 6A-6D. CaCC channel activity in the whole-cell configuration wasmeasured in HT-29 cells. FIG. 6A illustrates ionomycin-induced currentsin the absence or presence of CaCC_(inh)-A01 or CaCC_(inh)-B01 recordedat a holding potential at 0 mV, and pulsing to voltages between ±120 mVin steps of 20 mV. FIG. 6B is a current/voltage (IN) plot of meancurrents at the end of each voltage pulse as in A. FIG. 6C is a summaryof current density data measured at Vm of +100 mV (S.E., n=6-8). FIG. 6D(top) illustrates ionomycin-induced chloride currents in the whole-cellconfiguration with symmetrical NMDG-Cl solutions. FIG. 6D (bottom) showsthe current/voltage plot of mean currents at the end of each voltagepulse.

FIG. 7A-7B present data showing inhibition of chloride secretion in T84cells by short-circuit current analysis. In FIG. 7A, after carbacholstimulation, ATP-induced I_(sc) was measured in the absence or presenceof inhibitors (representative of 3 or more separate experiments). FIG.7B represents a summary of carbachol and ATP-induced short-circuitcurrent in the absence or presence of CaCC_(inh)-A01 or CaCC_(inh)-B01(S.E., n=3-4). *P_(<)0.05 vs. control.

DETAILED DESCRIPTION

The disclosure herein relates to identification of specific and potentcompounds that inhibit (i.e., block) calcium-activated chloride channels(CaCCs) such that chloride movement through these channels in inhibited.These compounds, and compositions comprising these compounds may beuseful for treating secretory diarrheas and for treating pulmonarydiseases and disorders that exhibit and are exacerbated by excess mucusproduction (i.e., mucus hypersecretion), such as asthma, cysticfibrosis, chronic obstructive pulmonary disease (COPD), andbronchiectasis. Also described herein are methods that were developedfor identifying such compounds that inhibit or block calcium-activatedchloride channels.

Calcium-activated chloride channels (CaCCs) are widely expressed inmammalian tissues, including intestinal and lung epithelia, where theyfacilitate fluid secretion. Fluid secretion is an important mechanismfor maintaining normal function of the lungs, and intestinal tract, aswell as other body organs, and is regulated, in part, by chloride (Cl⁻)movement across the cell membrane.

Aberrantly increased secretion of chloride ion and water from a lungcell or intestinal cell, for example, may be facilitated by increasedconductance of chloride ion through one or more CaCC(s). Excess chlorideand water secretion from intestinal epithelial cells occurs in secretorydiarrheas that may be caused by administration of particular drugs(e.g., antiretroviral drugs or chemotherapeutic drugs) and by variousviruses, bacteria, and toxins. Aberrantly increased (or abnormallyincreased) secretion of chloride ion and water from a cell, such as alung cell or intestinal cell, therefore refers to increased secretion ofchloride ion and water from a cell compared with the amount or level ofchloride ion and water secreted from a cell that exhibits normalfunction, such as in a subject who does not have or exhibit symptoms ofconditions, diseases, and disorders discussed herein, such as secretorydiarrhea and certain chronic lung diseases (e.g., cystic fibrosis, COPD,bronchiectasis, asthma).

Available compounds that inhibit CaCCs, including fenamates,anthracene-9-carboxylic acid, indoleacetic acid, ethacrynic acid, andtamoxifen, have low potency and inhibit multiple types of Cl⁻ channelsand transporters, and in some cases cause activation of BK_(Ca) K⁺channels (see, e.g., Hartzell et al., Annu. Rev. Physiol. 67:719-58(2005)). Despite the need for potent, selective CaCC inhibitors, nonehas yet been available (see, e.g., reviews by Kidd et al., supra;Hartzell et al., supra).

At least five distinct classes of mammalian Cl⁻ channels, including thecystic fibrosis transmembrane conductance regulator (CFTR), CLC-typevoltage-sensitive Cl⁻ channels, ligand-gated (GABA and glycine) Cl⁻channels, volume-sensitive Cl⁻ channels, and calcium-activated Cl⁻channels (CaCCs) have been identified (Hartzell et al., supra;Eggermont, Proc. Am. Thorac. Soc. 1:22-7 (2004)). The molecularidentities of epithelial cell CaCCs remain unclear. Recently, a memberof a group of plasma membrane proteins with previously unknown function,referred to as TMEM16A, has been identified as associated withcalcium-dependent chloride current (see, e.g., Schroeder et al., Cell134:1019-29 (2008); Caputo et al., Science 322:590-94 (2008); Yang etal., Nature 455:1210-15 (2008)). Potential candidates includebestrophins (Best1-Best4), CLCs of the CKCA family, and products of therecently described tweety gene (Suzuki, Exp. Physiol. 91:141-7 (2006);Hartzell et al., Physiol. (Bethesda) 20:292-302 (2005); Loewen et al.,Physiol. Rev. 85:1061-92 (2005); Evans et al. J. Biol. Chem.279:41792-800 (2004)). Agents that inhibit at least one CaCCs wouldtherefore also be useful for characterizing and identifying CaCCs.Specific compounds, such as the compounds described herein, may beuseful for identifying CaCCs physiologically and may be used to isolatespecific currents from a mixture of currents. Such compounds may also beuseful for characterizing the pore (e.g., resolving the structure of thepore), analyzing tissue and cell type distribution of CaCCs, and forbiochemical analysis and manipulation.

Methods for Identifying Calcium-Activated Chloride Channel Inhibitors

Methods are provided herein for identifying agents that are inhibitors(i.e., agents that inhibit, block, prevent, interfere with, abrogate, ordecrease activity or activation in a statistically or biologicallysignificant manner) of at least one (i.e., one or more) CaCCs, therebyinhibiting movement of an anion, particularly chloride, from a cell(i.e., efflux or secretion from the cell). Thus, agents identified inthe methods described herein (including the aminothiophene andaminothiazole compounds described herein) inhibit chloride conductance(or current) through a CaCC. The methods also include high throughputformats that are useful for screening large numbers of candidate agentsto identify agents that are CaCC inhibitors.

Provided herein are methods to identify inhibitors (e.g., small moleculeinhibitors) of one or more mammalian (particularly human) CaCCs and thatthereby inhibit chloride and/or water secretion from a cell. The methodsmay be used to identify inhibitors that inhibit the channel itself(i.e., bind to or block the CaCC) or that inhibit by targeting a site orsites distal to calcium elevation. In a certain specific embodiment, acell-based assay is provided that determines iodide influx in amonolayer of yellow fluorescent protein (YFP)-expressing cells (such asmammalian epithelial cells) in response to one or more calcium-elevatingagonists. In certain embodiments, the methods described herein may alsoinclude agonists that prevent or minimize identification of compoundsthat inhibit at a target site or sites proximal to cell calciumelevation.

In certain embodiments, methods described herein for identifying anagent that inhibits a CaCC (i.e., that inhibits activation of a CaCC)and methods for determining anion influx include iodide as the anion.Iodide is useful in the methods described herein, particularly ininitial screening of large numbers of agents because iodide istransported by channels, as opposed to being transported by eitherexchangers or cotransporters, such as AE1 (Cl⁻/HCO₃ ⁻) and NKCC(Na⁺/K⁺/2Cl⁻). In addition, compared to chloride, iodide demonstratesgreater quenching compared with presently available molecular andchemical halide sensors (see, e.g., Verkman et al., Methods Mol. Med.70:187-96 (2002); Hartzell et al., Annu. Rev. Physiol. 67:719-58 (2005);Eggermont, Proc. Am. Thorac. Soc. 1:22-7 (2004); each of thesereferences are hereby incorporated by reference in their entirety).Accordingly, as additional chemical and macromolecular halide sensorsbecome available, additional halides may be used in these methods todetermine anion flux.

In one embodiment, methods for identifying an agent that is an inhibitorof a calcium-activated chloride channel comprise contacting (i.e.,combining in some manner that permits interaction between components inthe method) a cell, particularly such as an epithelial cell, and acandidate agent to provide a test sample (i.e., mixture, combination),under conditions and for a time sufficient to permit interaction betweenthe candidate agent and the cell, wherein the cell comprises acalcium-activated chloride channel and also comprises a cytoplasmicindicator protein that binds halide. To the test sample of the cells andthe candidate agent is added at least one calcium-elevating agonist anda halide (e.g., Cl⁻, I⁻, Br⁻, or F⁻) or NO₃ ⁻ (i.e., a source of halideor NO₃ ⁻) other than chloride, such as iodide. In certain embodiments,the candidate agent is added prior to contacting the cells with the atleast one calcium-elevating agent and a halide or NO₃ ⁻. In otherembodiments, the candidate agent is added contemporaneously with orsubsequently to the at least one calcium-elevating agonist and a halideor NO₃ ⁻. The halide or NO₃ ⁻ is in a solution that lacks chloride(e.g., a phosphate buffered iodide solution). The at least onecalcium-elevating agonist is added in an amount sufficient that uponbinding to or interacting with the cell, the level of intracellularcalcium ion (Ca²⁺) increases in the cell, and results in activation(i.e., opening) of a calcium-activated chloride channel. In the absenceof an inhibitor of the CaCC, activation of the channel results inincreased influx of the anion (e.g., iodide). The capability of thecandidate agent to inhibit influx of the anion is determined bydetermining and then comparing the level of anion influx in the presenceand the level of anion influx in the absence of the candidate agent. Adecrease in the level of anion influx in the presence of the candidateagent compared with the level of anion influx in the absence of thecandidate agent indicates that the candidate agent inhibits the movementof the anion (i.e., inhibits anion current or conductance of the anionthrough the channel). As described in greater detail herein, the levelof anion influx may be determined by determining the level of binding ofthe anion (e.g., iodide) to the cytoplasmic indicator protein. Anexemplary cytoplasmic indicator protein is a halide sensor chromophoresuch as yellow fluorescent protein, or a mutant (i.e., variant) thereof,that upon binding to or interacting with the anion, the fluorescentsignal is quenched (i.e., decreased). The change in the fluorescentsignal may be determined at multiple time points over a time course(i.e., length of time) to provide time course data for determining anioninflux. In a certain embodiment, the cells are epithelial cells, and incertain specific embodiments, the epithelial cells endogenously expressat least one CaCC. In other embodiments of any one of the methodsdescribed herein, a cell (such as an epithelial cell) may betransfected, transformed, or transduced with a recombinant expressionvector that comprises a polynucleotide that encodes a CaCC as discussedin further detail herein.

As discussed in greater detail herein, a test sample may be containedwithin any one of a variety of vessels that can include cells, buffer,and/or media, and components of the assay. By way of example, the cellsmay be adherent cells that are attached to (or adhered to) a well of acell culture plate. The differing components (e.g., candidate agent(s),calcium-elevating agonist(s), halide, NO₃ ⁻, etc.) used in the methodsdescribed herein may be contacted with the cells by addition of acomponent to a solution, or as a solution, in which the cells are bathed(i.e., supernatant). Each of the steps of the methods described hereinare performed under conditions and for a time sufficient appropriate foreach step. Such conditions and time are discussed herein and in theexemplary methods provided in the examples, and which may be readilydetermined by persons skilled in the art.

As described in greater detail below, the cell (e.g., an epithelialcell) comprising the cytoplasmic indicator protein is obtained bytransforming, transfecting, or transducing the epithelial cell with arecombinant expression vector that comprises a polynucleotide thatencodes the cytoplasmic indicator protein.

In a more specific embodiment, a method for identifying an agent that isan inhibitor of a calcium-activated chloride channel (CaCC) comprisescontacting (i.e., combining in some manner that permits interactionbetween components in the method) an epithelial cell, and a candidateagent to provide a test sample (or combination or mixture), underconditions and for a time sufficient to permit interaction between thecandidate agent and the epithelial cell, wherein the epithelial cellcomprises a calcium-activated chloride channel and also comprises acytoplasmic indicator protein that binds a halide, such as iodide.Subsequent to a time sufficient for the epithelial cells and agent tointeract, the test sample (or combination or mixture) of the epithelialcells and the candidate agent are then contacted with at least onecalcium-elevating agonist and iodide under conditions and for a timesufficient for the calcium-elevating agonist and epithelial cell tointeract. Iodide is added to the test sample containing epithelial cellsin the absence of a source of chloride (e.g., for example, sodiumchloride is not included in buffers, media, or other reagents). Anexemplary source of iodide is a phosphate buffer comprising iodide.Iodide may be added at concentrations between 10 mM and 200 mM dependingupon the combination of cells, calcium-elevating agonist(s), agents,CaCC(s) expressed by the cells, and other components and conditions(such as media) that are used in the method. The at least onecalcium-elevating agonist is added in an amount sufficient that uponbinding or interacting to the cell, the level of intracellular calciumion (Ca²⁺) increases in the cell, causing activation (i.e., opening) ofa calcium-activated chloride channel. In the absence of an inhibitor ofthe CaCC, activation of the channel results in increased influx ofiodide. The capability of the candidate agent to inhibit influx ofiodide is determined by determining and then comparing the level ofiodide influx in the presence of the candidate agent and the level ofiodide influx in the absence of the candidate agent. A decrease in thelevel of iodide influx in the presence of the candidate agent comparedwith the level of iodide influx in the absence of the candidate agentindicates that the candidate agent inhibits the movement of the iodide(i.e., inhibits anion current or conductance of the anion through thechannel).

As described above, the level of anion (e.g., iodide) influx may bedetermined by determining (i.e., in some manner measuring) the level ofbinding of iodide to the cytoplasmic indicator protein. An exemplarycytoplasmic indicator protein is a halide sensor chromophore such asyellow fluorescent protein, or a mutant or variant thereof, that uponbinding or interaction of iodide, the fluorescent signal is quenched(i.e., decreased). The change in the fluorescent signal may bedetermined at multiple time points to provide time course data fordetermining iodide influx. The fluorescent signal can be detected by anyone of numerous commercially available apparatus.

Calcium-activated chloride channels are activated by cytosolic calciumion (Ca²⁺). The Ca²⁺ that activates a CaCC may come from either Ca²⁺influx or from Ca²⁺ release from intracellular stores. Without wishingto be bound by any particular theory, a CaCC may be activated by directCa²⁺ binding or may act indirectly on the CaCC by binding to Ca²⁺binding proteins or Ca²⁺ dependent enzymes. Certain CaCCs may bestimulated by protein phosphorylation that involvescalcium-calmodulin-dependent kinase II. Experimentally, cytosolic Ca²⁺may be induced by stimulating cells with Ca²⁺ elevating agonists and byCa²⁺ ionophores, such as ionomycin (see, e.g., Eggermont, supra; Kidd etal., supra). Exemplary calcium-elevating agonists that may be used inthe methods described herein include but are not limited to histamine,calcimycin, ATP, carbachol, and forskolin. Without wishing to be boundby any particular theory, the calcium elevating agonist binds to acognate receptor on the cell, which triggers phosphoinositide signaling,which in turn activates calcium-calmodulin-dependent kinase II. Thecognate receptor of the calcium-elevating agonist, carbachol, is amuscarinic cholinergic receptor (m₃AchR). ATP, acting as acalcium-elevating agonist binds to purinergic receptor (P_(2Y2)). Incertain embodiments, one calcium-elevating agonist is contacted with thecells (e.g., epithelial cells). In a specific embodiment, thecalcium-elevating agonist is carbachol or ATP. In other certainembodiments, at least two calcium-elevating agonists are contacted withthe cells. In certain other embodiments, at least three or at least fourcalcium-elevating agonists are included in the methods described herein.In a specific embodiment, at least two calcium-elevating agonists, forexample, carbachol and ATP, are contacted with the cells in a mannerthat permits interaction between the cells and each of the agonists.

Whether a single agonist or multiple agonists (i.e., at least two,three, or four agonists) are used in a screening assay for identifyingCaCC inhibitors may depend on the particular cell (including theparticular epithelial cell or cell line) that is used in the screeningmethod. Selection of one or more appropriate calcium-elevating agonistscan be determined by using the methods described herein for determininganion influx in a cell (e.g., an epithelial cell) in the presence andabsence of the agonist. The concentration of the one or morecalcium-elevating agonists that are contacted with cells to elevatecytosolic Ca²⁺ can be determined according to methods routinelypracticed by a skilled artisan when optimizing assay methods; typicallya calcium-elevated agonist is added to cells (e.g., epithelial cells) ata concentration between 10-100 μM.

A person skilled in the art also readily appreciates that appropriatecontrols can be designed and included when performing the in vitromethods described herein. For example, a method for identifying an agentthat inhibits a CaCC and that inhibits influx of an anion (e.g., iodide)includes a sample or samples that comprise a plurality of cells (e.g.,epithelial cells) and one or more candidate agents and includes one ormore control samples. Controls may include samples that are used todetermine the basal iodide conductance (or the basal level of anotherhalide or nitrate conductance for which influx is determined), which isthe conductance observed in the absence of a calcium-elevated agonist,which is the calcium-chloride channel-independent conductance.Additional controls that may be included in the methods for identifyingan agent are samples that comprise the vehicle (i.e., solvent, buffer,or solution) in which the agent is prepared but lacks the agent(negative control). Instead of or in addition to the aforementionednegative control, a sample may include an agent or compound that isknown not to inhibit CaCC and not to alter anion flux. Accordingly,determining anion (e.g., iodide) flux in the presence and absence of anagent is understood to mean that a first sample comprises all assaycomponents (including the agent) and at least one additional sample(i.e., a second, third, fourth, and/or fifth sample etc.) comprises allcomponents except the agent, respectively. The composition of controlsamples (i.e., including (in the presence of) or excluding (in theabsence of) certain components) and the time at which the controlsamples are prepared and evaluated are described herein and can also bereadily determined by a person skilled in the assay method art.Conditions for a particular assay include temperature, buffers(including salts, cations, media), and other components that maintainthe integrity of the cell, the agent, the calcium-elevating agonist,with which a person skilled in the art will be familiar and/or which canbe readily determined. Analysis of positive and negative controls may beincluded in any method prior to, concurrently with, or subsequent todetermining the ability of an agent to inhibit CaCC(s).

Reference to “an agent” or “a candidate agent” includes a plurality ofsuch agents. Accordingly, as described herein a sample comprising cells(e.g., epithelial cells) and an agent may comprise one, two, three,four, or any number of agents between 1 and 10 or 1 and 50. As routinelypracticed in the art for screening libraries of agents, multiple agentsmay be included in a single sample, the method performed, positive“hits” selected, and a subsequent assay performed in which a lessernumber of agents are included per sample. The methods are repeated untilthe method is performed with a single agent per sample.

With respect to the methods discussed above and herein, reference to “acell” is not necessarily limited to a single cell but is intended toinclude at least one cell (i.e., one or more cells) or a plurality ofcells. Reference to “contacting” in the steps of the methods describedherein includes incubating, immersing, exposing, bathing, combining,mixing, adding together, or otherwise introducing one component (e.g., acell, candidate agent, halide (e.g., iodide) or NO₃ ⁻, agonist such as acalcium-elevating agonist, or any other component described herein) ofthe method with another component. The steps of the methods describedherein are performed under conditions and for a time sufficient

As used herein, movement of a halide, such as chloride or iodide orother anion, across and through the outer cell membrane from theextracellular space or environment into the cell refers to influx of thehalide. Movement of a halide, such as chloride or iodide or other anion,out of the cell into the extracellular space refers to efflux of thehalide.

As described herein, the cells (e.g., epithelial cells) comprise atleast one CaCC. Other chloride channels (e.g., cystic fibrosistransmembrane conductance regulator protein (CFTR); CLC-typevoltage-sensitive chloride channels; ligand gated (GABA and glycine)chloride channels; and volume-sensitive channels) may be expressed inthe cell in addition to one or more CaCCs. The methods described herein,may therefore, further comprise contacting the cells (e.g., epithelialcells) with one or more bioactive agents that inhibit one or morechloride channels but that do not inhibit CaCCs. For example, certaincells, including epithelial cells and epithelial cell lines, may expresscystic fibrosis transmembrane conductance regulator protein (CFTR) inaddition to at least one CaCC. Accordingly, an inhibitor of CFTR may becontacted with the cells to block or inhibit transport of chloride orother halide through CFTR. Exemplary inhibitors of CFTR include, but arenot limited to thiazolidinone compounds (e.g.,3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTR_(inh)-172) and hydrazide compounds (see,e.g., U.S. Pat. No. 7,235,573; U.S. Patent Application Publication No.2005-0239740; Muanprasat et al., J. Gen. Physiol. 124:125-37 (2004); Maet al., J. Clin. Invest. 110:1651-58 (2002)).

The methods described herein have value in high throughput screening,that is, in automated screening of a large number of candidate agentsthat inhibit calcium-activated chloride channels and that are thususeful for inhibiting chloride secretion from a cell. The method may beused to screen synthetic or natural product libraries for bioactiveagents and compounds. The methods described herein are thereforeamenable to automated, cost-effective high throughput drug screening andhave immediate application in a broad range of pharmaceutical drugdevelopment programs. In one embodiment, the agents to be screened areorganized in a high throughput screening format such as usingmicrofluidics-based devices, or a 96-well plate format, or other regulartwo dimensional array, such as a 384-well, 48-well or 24-well plateformat, or an array of test tubes. The format is therefore amenable toautomation. An automated apparatus that is under the control of acomputer or other programmable controller may be used for one or moresteps of the methods described herein. A controller can monitor theresults of each step of the process and can automatically alter thetesting paradigm in response to those results.

For high-throughput screening, the cells, such as epithelial cells,comprising at least one CaCC may be cultured and used in the methodsdescribed herein in any of a variety of containers or sample vessels,including test tubes, multi-well plates such as 48-well, 72-well,96-well plates, 384-well plates or other such vessels, including thoseuseful for high throughput screening formats wherein, for example,detection of fluorescence of the cytoplasmic indicator protein in aplurality or reaction vessels, may be automated. Epithelial cells aretypically adherent cells in culture, and the surface to which the cellsare adhered may be solid, such as a tissue culture plate (e.g., 24-well,48-well, 72-well, 96-well plates, 384-well plate), or the cells may beadhered to microcarrier beads. Alternatively, the surface on which thecells adhere may be porous such that the apical cell surface andbasolateral cell surface may be exposed to or bathed in the solutionsdescribed herein.

The number of samples to be assayed may influence the degree ofautomation that can be implemented. For example, when high throughputscreening, (i.e., assaying a large number of samples in a relativelybrief time period) is desired, robotic or semi-robotic instruments maybe used. In certain instances, microfluidics multiplexing technologiesmay be employed (see, e.g., Thorsen et al., Science 298:580-84 (2002);Manz and Becker, eds. Microsystem Technology in Chemistry and LifeSciences (Springer 1999); Zhang et al, Microelectrofluidic Systems:Modeling and Simulation (CRC Press 2002); Tabeling, Introduction toMicrofluidics (Oxford University Press 2006); U.S. Pat. Nos. 6,969,850;6,878,755; 6,454,924; 6,681,788; 6,284,113). Alternatively, samples maybe processed manually, even for formats that accommodate large samplenumbers (e.g., 96-well microplates).

In a specific embodiment, a high throughput method is provided foridentifying an agent that is an inhibitor of a calcium-activatedchloride channel. In one embodiment, the method comprises culturing, ineach of a plurality of reaction vessels in a high throughput screeningarray, a plurality of cells (e.g., epithelial cells), wherein theplurality of cells comprise (a) a calcium-activated chloride channel and(b) a cytoplasmic indicator protein that binds halide (and may also bindNO₃ ⁻). As described above and herein, the plurality of cells in each ofthe plurality of reaction vessels, are contacted (i.e., combined with orin some manner permitted to interact with) a candidate agent underconditions and for a time sufficient to permit interaction between thecandidate agent and the plurality of the cells, to form a test sample(or combination or mixture) in each of the plurality of reactionvessels. Subsequently added to each test sample in each reaction vesselis at least one calcium-elevating agonist and a halide or NO₃ ⁻, underconditions and for a time sufficient for the calcium-elevating agonistto bind to the plurality of cells in each of the plurality of reactionvessels. In certain particular embodiments, the halide is iodide. Anyone, two, or more of the calcium-elevating agonists described hereininteracts with the plurality of cells for a time sufficient to increasethe level of calcium ion (Ca²⁺) in the plurality of cells. The level ofhalide (e.g., iodide) or NO₃ ⁻ influx in the plurality of epithelialcells in each of the plurality of reaction vessels in the presence ofthe candidate agent is compared with the level of halide (e.g., iodide)or NO₃ ⁻ influx in the absence of the candidate agent. A decrease in thelevel of halide (e.g., iodide) or NO₃ ⁻ influx in the presence of thecandidate agent compared with the level of halide (e.g., iodide) or NO₃⁻ influx in the absence of the candidate agent, indicates that thecandidate agent is an inhibitor of the calcium-activated chloridechannel. In a certain embodiment, the cells are epithelial cells, and incertain specific embodiments, the epithelial cells endogenously expressat least one CaCC.

Also provided herein are methods for determining anion (i.e., halide andnitrate (NO₃ ⁻)) transmembrane movement (i.e., conductance or current)through calcium-activated chloride channels (CaCCs). These methods maybe used to determine if a particular cell comprises a CaCC or toquantify the level of a CaCC expressed by the cell. The methods may alsobe useful for identifying and characterizing a CaCC that is endogenouslyor exogenously expressed by a cell. For example, for exogenousexpression, a recombinant expression vector that comprises apolynucleotide (which polynucleotide is operatively linked to at leastone expression control region, such as a promoter) that encodes apolypeptide believed to be or known to be a CaCC may be introduced intoa cell by transformation, transfection, or transduction of a cell, suchas an epithelial cell or other cell type, according to molecular biologyand protein expression methods routinely practiced by person skilled inthe art and described in detail herein (see, e.g., Ausubel et al.(Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & JohnWiley & Sons, Inc., 1993)); Sambrook et al. (Molecular Cloning: ALaboratory Manual, 3rd Ed., (Cold Spring Harbor Laboratory 2001));Maniatis et al. (Molecular Cloning, (Cold Spring Harbor Laboratory1982); see also discussion below with respect to cells that may be usedin the methods described herein). The methods may also be useful foridentifying and characterizing calcium-elevating agonists, singly or incombination, that effectively increase the level of calcium ion in thecell such that the calcium-activated CaCC(s) is activated and opens,permitting movement of anions through the channel.

In one embodiment, influx of an anion (such as a halide (Cl⁻, I⁻, Br⁻,or F⁻) or NO₃) in a cell, for example, an epithelial cell, thatcomprises or is suspected of comprising at least one CaCC (i.e., one ormore CaCCs), may be determined by contacting (i.e., combining in somemanner that permits interaction) the anion, the cell, and acalcium-elevating agonist under conditions and for a time sufficientthat permit the calcium-elevating agonist to interact with the cell.Anion influx in the presence and absence of the calcium-elevatingagonist is then determined. The cell comprises a cytoplasmic indicatorprotein that is capable of interacting with an anion (such as a halide(Cl⁻, I⁻, Br⁻, or F⁻) or NO₃ ⁻) and thus acts as an anion sensor. Asdescribed above and herein, in particular embodiments, the anion isiodide (I⁻).

In a more specific embodiment, a method is provided for determining(i.e., measuring or quantifying) influx of an anion, which is a halide(Cl⁻, I⁻, Br⁻, or F⁻) or NO₃ ⁻, in epithelial cells that comprise or aresuspected of comprising at least one CaCC (i.e., one or more CaCCs). Theepithelial cells, which comprise a cytoplasmic indicator protein thatbinds to halides and NO₃ ⁻ described in greater detail herein, arecontacted with the anion and with a calcium-elevating agonist underconditions and for a time sufficient to permit the agonist to interactwith the epithelial cell. Anion influx in the presence and absence of acalcium-elevating agonist is then compared. As described above andherein, in particular embodiments, the anion is iodide (I⁻).

As described in greater detail below, the cell (e.g., an epithelialcell) comprising the cytoplasmic indicator protein is obtained bytransforming, transfecting, or transducing the epithelial cell with arecombinant expression vector that comprises a polynucleotide thatencodes the cytoplasmic indicator protein.

Cells.

As described herein, methods are provided for determining influx of ananion through a CaCC that traverses the outer cell membrane of the cell,such as an epithelial cell, and for determining the capability of anagent to inhibit influx of the anion through the activated CaCC into thecell. As described in detail herein, epithelial cells may be used as thecells that comprise at least one CaCC and that comprise a cytoplasmicindicator protein in the methods for determining anion influx in thepresence of a calcium-elevating agonist and in the methods foridentifying an agent that inhibits anion influx in a cell. Desirablecharacteristics of the cells include the capability of the cells to growefficiently on a solid support (particularly a plastic support) (e.g.,cell culture plates and flasks routinely used for cell culture methodsand reaction vessels that are used in high throughput screeningmethods); capability to remain adhered to the support during repeatedchanges of media and washing; efficient expression of the cytoplasmicindicator protein to provide a measurable difference in the signal(e.g., fluorescence) detected prior to anion influx and the signaldetected after anion influx (e.g., influx of iodide) that can beprecisely and accurately determined; efficient and strong CaCC chlorideconductance (i.e., chloride current or movement through a CaCC); and lowbasal (i.e., CaCC-independent) conductance of the anion (e.g., iodide).Exemplary epithelial cells as described herein include HT-29 intestinalepithelial cells that, after infection with a lentiviral vector thatencoded the cytoplasmic indicator protein YFP-H148Q/I152L, were brightlyfluorescent and able to grow in confluent monolayers in culture.

Cells, such as epithelial cells, that may be used in the methodsdescribed herein may endogenously or exogenously express at least oneCaCC. Cells that exogenously express a CaCC may be prepared according tomolecular biology and protein expression methods routinely practiced inthe art. For example, for exogenous expression, a recombinant expressionvector that comprises a polynucleotide (which polynucleotide isoperatively linked to at least one expression control region, such as apromoter) that encodes a polypeptide believed to be or known to be aCaCC may be introduced into a cell by transformation, transfection, ortransduction of a cell, such as an epithelial cell or other cell type,according to molecular biology and protein expression methods routinelypracticed by person skilled in the art and described in detail herein.Cell lines may be established that stably express the exogenouslyintroduced CaCC, or cells may be used in the methods described hereinthat transiently express the CaCC.

By way of example, such an expression vector that includes apolynucleotide comprising a nucleotide sequence that encodes a CaCCreferred to in the art as TMEM16A may be introduced into a cell, bymolecular biology methods routinely practiced in the molecular biologyart (see, e.g., Ausubel et al. (Current Protocols in Molecular Biology(Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., 1993)); Sambrook etal. (Molecular Cloning: A Laboratory Manual, 3rd Ed., (Cold SpringHarbor Laboratory 2001)); Maniatis et al. (Molecular Cloning, (ColdSpring Harbor Laboratory 1982)). The TMEM16A CaCC may be of mammalianorigin (such as mouse, human, rat) or may be from a non-mammalianspecies or may be from algae. Polynucleotide sequences and the encodedpolypeptide sequences for TMEM16A of different animals and plants arereadily available from public databases. In certain embodiments, apolynucleotide encoding human TMEM16A (see, e.g., GenBankNM_(—)018043.5)may be included in a recombinant expression vector wherein the encodingpolynucleotide is operatively linked to at least one expression controlsequence (e.g., a promoter). See also, for example, Schroeder et al.,Cell 134:1019-29 (2008); Caputo et al., Science 322:590-94 (2008); Yanget al., Nature 455:1210-15 (2008)) (all of which are incorporated hereinby reference in their entirety).

For maintaining viability of cells, including epithelial cells, thecells are cultured in media and under conditions practiced in the artfor proper maintenance of cells in culture, including media (with orwithout antibiotics) that contains buffers and nutrients (e.g., glucose,amino acids (e.g., glutamine), salts, minerals (e.g., selenium)) andalso may contain other additives or supplements (e.g., fetal bovineserum or an alternative formulation that does not require a serumsupplement; transferrin; insulin; putrescine; progesterone) that arerequired or are beneficial for in vitro culture of cells and that arewell known to a person skilled in the art (see, for example, GIBCOmedia, INVITROGEN Life Technologies, Carlsbad, Calif.). Similar tostandard cell culture methods and practices, the cell cultures describedherein are maintained in tissue culture incubators designed for such useso that the levels of carbon dioxide (typically 5%), humidity, andtemperature can be controlled. The cell culture system may also compriseaddition of exogenous (i.e., not produced by the cultured cellsthemselves) cell growth factors, which may be provided, for example, inthe media or in a substrate or surface coating. Growth characteristicsof the cells for use in the methods described herein, may be optimizedby altering the composition or type of media, adjusting the amount ofone or more nutrients and/or serum, which are procedure with which askilled artisan is familiar. Persons skilled in the tissue culture artalso recognize that conditions employed for routine maintenance of acell culture (i.e., media, additives, nutrients) may need to be adjustedappropriately for certain manipulations of the cells (for example,successful introduction of a recombinant expression vector (plasmid orviral vector, including a retroviral vector); ensuring appropriateconfluency and growth properties of cells for high throughputscreening). By way of example, HT-29 cells are typically cultured inMcCoy's 5a medium (see ATCC recommendation). After transduction with alentiviral vector that comprises a polynucleotide that encodes thecytoplasmic indicator protein, the cells divide more rapidly androbustly in a different media, such as Dulbecco's Modified Eagle'sMedium (DMEM).

In certain embodiments, the cell is epithelial cell, which is anintestinal epithelial cell; in other certain embodiments, the epithelialcell is a lung epithelial cell. Epithelial cells and other cell typesmay be obtained of derived from any one of a number of animals,including mammals. Mammalian cells may be obtained from humans;non-human primates; rodents such as mice, rats, or rabbits; cats(feline); dogs (canine); cattle (bovine); sheep (ovine); pigs (porcine);llamas; and camels, for example. The cells may be derived from a primarycell culture (e.g., lung epithelial cells or intestinal epithelial cellsor other cells that endogenously express a CaCC), or culture adaptedcell line, including but not limited to, genetically engineered celllines that may contain chromosomally integrated or episomal recombinantnucleic acid sequences, immortalized or immortalizable cell lines,somatic cell hybrid cell lines, differentiated or differentiatable celllines, transformed cell lines, and the like. Exemplary intestinalepithelial cells that may be used in the methods described herein areHT-29 cells (American Type Culture Collection, Manassas, Va.).

Cytoplasmic Indicator Protein.

The cells that are used in the methods described herein, includingepithelial cells such as HT-29 intestinal epithelial cells, comprise atleast one CaCC that traverses the outer cell membrane of the cell. Thecells further comprise a cytoplasmic indicator protein that is a halidesensor. In a specific embodiment, the cytoplasmic indicator protein is achromophore, such as the green fluorescent protein variant (i.e., amutant), called yellow fluorescent protein (e.g., YFP-H148Q) (see, e.g.,Jayaraman et al. J. Biol. Chem. 275:6047-50 (2000); Galietta et al., Am.J. Physiol. Cell Physiol. 281:01734-42 (2001); Ma et al., J. Clin.Invest. 110:1651-58 (2002); Muanprasat et al., J. Gen. Physiol.124:125-37 (2004); Ma et al. J. Biol. Chem. 277:37235-41 (2002); Yang etal., J. Biol. Chem. 278:35079-85 (2003); Ormo et al., Science273:1392-95 (1996); Elsliger et al., Biochemistry 38:5296-301 (1999);Wachter et al., Structure (Lond. 6:1267-77 (1998); Wachter et al., Curr.Biol. 9:R628-29 (1999); Pedemonte et al., J. Clin. Invest. 115:2564-71(2005)). In certain specific embodiments, the cytoplasmic indicatorprotein is a yellow fluorescent protein mutant (i.e., variant) in whichone or more additional amino acid have been substituted that conferparticular desirable properties (for example, altering the level offluorescence and/or altering the binding affinity of the yellowfluorescent protein for one or more halides). In a particularembodiment, a YFP mutant (i.e., variant), YFP-H148Q/I152Q, may be usedfor the methods described herein to identify an agent that inhibitsCaCC(s) or for the methods to determine anion flux in a cell (see, e.g.,Galietta et al., FEBS Lett. 499:220-24 (2001); Yangthara et al., Mol.Pharmacol. 72:86-94 (2007)). The YFP-H148Q/I152Q indicator protein hasiodide and nitrate (NO3⁻) sensitivities, which permit measurement ofcellular halide movement by chloride/nitrate exchange or bychloride/iodide exchange using low iodide concentrations.

Epithelial cells, such as HT-29 cells, expressing a cytoplasmicindicator protein that is a yellow fluorescent protein (YFP), or mutant(i.e., variant) thereof (e.g., YFP-H148Q/I152Q) are brightly fluorescent(see FIG. 1). When an extracellular halide, such as iodide, is contactedwith or added to cells that express a CaCC and a cytoplasmic indicatorprotein such as the yellow fluorescent protein, or mutant (i.e.,variant) thereof, in the presence of at least one calcium-elevatingagonist, influx of an extracellular halide is facilitated by a CaCC.Influx of the halide, such as iodide, is detected by fluorescentquenching of the YFP halide sensor (see the schematic in FIG. 1A). Thusin certain embodiments, iodide influx is determined by measuringfluorescence quenching of a cytoplasmic YFP-based halide sensor(YFP-H148Q/I152L).

Fluorescence may be quantified using any one of a number of fluorescencedetection systems, including “plate readers” that detect the signal inindividual wells of a multi-well plate, available from commercialvendors. The YFP indicator protein (YFP-H148Q/I152L) is approximately50% quenched by approximately 3 mM iodide. As discussed herein, iodidemay be added at concentrations between 10 mM and 200 mM depending uponthe combination of cells, calcium-elevating agonist(s), agents, CaCC(s)expressed by the cells, and other components and conditions (such asmedia) that are used in the method.

For transient or stable expression of a cytoplasmic indicator protein(e.g., the YFP mutants (i.e., variants) thereof described herein, forexample, YFP-H148Q/I152Q), cells that comprise at least one CaCC(endogenously or exogenously) may be transformed, transfected, ortransduced with a recombinant expression vector that comprises apolynucleotide that encodes the cytoplasmic indicator protein. Plasmidsthat encode YFP mutants (i.e., variants) may be obtained from commercialsources (e.g., CLONTECH, Mountain View, Calif.) (for example,EYFP=GFP-S65G/V68L/S72A/T203Y). Additional substitutions of amino acids,such as substitution of histidine at position 148 with glutamine (H148Q)and substitution of isoleucine at position 152 with glutamine (I152Q)may be performed by site-directed mutagenesis methods routinely andcommonly practiced by persons skilled in the art (see, e.g., Galietta etal. FEBS Lett. 499:220-24 (2001); Ausubel et al. (Current Protocols inMolecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc.,1993)); Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rdEd., (Cold Spring Harbor Laboratory 2001)).

In a specific embodiment of the methods described herein, cells, such asepithelial cells, including intestinal epithelial (e.g., the exemplarycell line, HT-29) and lung epithelial cells or epithelial cells derivedfrom other tissues, comprise an exogenous polynucleotide that encodesthe cytoplasmic indicator protein (e.g., YFP-H148Q/I152Q). The cells,(e.g., epithelial cells) may be transfected, transformed, or transducedwith a recombinant expression vector, which comprises a polynucleotidethat is capable of directing expression of the cytoplasmic indicatorprotein. To direct expression of the cytoplasmic indicator protein, thepolynucleotide comprises a nucleotide sequence that encodes thecytoplasmic indicator protein, which nucleotide sequence is operativelylinked to at least one expression control sequence (e.g., a promoter,enhancer, transcriptional control element, and the like). Recombinantexpression vectors may be prepared according to methods and techniqueswith which a person skilled in the molecular biology art is familiar andwhich are described herein.

Cells (e.g., epithelial cells) containing the described recombinantexpression constructs may be genetically engineered (transduced,transformed, or transfected) with vectors and/or expression constructs(for example, a cloning vector, a shuttle vector, or an expressionconstruct). The vector or construct may be in the form of a plasmid,viral vector (which includes a viral particle), a phage, etc. Theengineered cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants, oramplifying particular genes or encoding-nucleotide sequences. Forparticular types of cells and particular cell lines, selection andmaintenance of culture conditions such as temperature, pH and the like,will be readily apparent to the ordinarily skilled artisan. Preferablythe cells, including epithelial cells, can be adapted to sustainedpropagation in culture to yield a cell line according to art-establishedmethodologies. In certain embodiments, the cell line is an immortal cellline, which refers to a cell line that can be repeatedly passaged inculture (at least ten times while remaining viable) following log-phasegrowth. In other embodiments the cell that is used to generate a cellline is capable of unregulated growth, such as a cancer cell, or atransformed cell, or a malignant cell.

Useful recombinant expression constructs are prepared by inserting intoan expression vector a structural DNA sequence encoding the polypeptideof interest, such as a cytoplasmic indicator protein or a CaCC, togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The construct may comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector construct and, if desirable, to provideamplification within the cell (e.g., an epithelial cell). A particularplasmid or vector may be used as long as it is replicable and viable inthe cell. Thus, for example, the polynucleotides that encode acytoplasmic indicator protein may be included in any one of a variety ofexpression vector constructs for expressing a polypeptide.

An appropriate DNA sequence(s) may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. Numerous standard techniques are described, forexample, in Ausubel et al. (Current Protocols in Molecular Biology(Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., 1993)); Sambrook etal. (Molecular Cloning: A Laboratory Manual, 3rd Ed., (Cold SpringHarbor Laboratory 2001)); Maniatis et al. (Molecular Cloning, (ColdSpring Harbor Laboratory 1982)), and elsewhere.

The nucleotide sequence encoding a protein of interest (e.g., acytoplasmic indicator protein or a CaCC) in the expression vector isoperatively linked to at least one appropriate expression controlsequence (e.g., a promoter or a regulated promoter) to direct mRNAsynthesis. Representative examples of such expression control sequencesinclude LTR or SV40 promoter, the E. coli lac or trp, the phage lambdaP_(L) promoter, and other promoters known to control expression of genesin prokaryotic or eukaryotic cells or their viruses. Promoter regionscan be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retroviruses, and mousemetallothionein-I. Selection of the appropriate vector and promoter andpreparation of certain recombinant expression constructs comprising atleast one promoter or regulated promoter operatively linked to apolynucleotide described herein is well within the level of ordinaryskill in the art.

In certain embodiments, the recombinant expression construct thatencodes the polypeptide of interest (e.g., a cytoplasmic indicatorprotein or a CaCc) is a retroviral vector, which may be a lentiviralvector. For example, retroviruses from which the retroviral vectors maybe derived include, but are not limited to, alpharetroviruses,betaretroviruses, gammaretroviruses, deltaretroviruses,epsilonretroviruses, lentiviruses such as the lentiviral vectorsdescribed in U.S. Pat. Nos. 5,981,276 and 6,312,682, in addition tospumavirusus. Specific examples of retroviruses that may be usedinclude, but are not limited to, Rous sarcoma virus, avian leukosisvirus, avian myeloblastosis virus, mouse mammary tumor virus, felinesarcoma virus, avian reticuloendotheliosis virus, myeloproliferativesarcoma virus, various murine leukemia viruses, Moloney murine leukemiavirus, Harvey murine sarcoma virus, bovine leukemia virus,T-lymphotropic viruses, avian leukosis virus, gibbon ape leukemia virus,feline leukemia virus, human immunodeficiency virus, simianimmunodeficiency virus, feline immunodeficiency virus, and bovineimmunodeficiency virus.

A viral vector also includes one or more promoters. Suitable promotersthat may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller et al., Biotechniques 7:980-990 (1989), or any otherpromoter (e.g., eukaryotic cellular promoters including, for example,the histone, pol III, and β-actin promoters). Other viral promoters thatmay be employed include, but are not limited to, adenovirus promoters,ubiquitin promoters, thymidine kinase (TK) promoters, and B19 parvoviruspromoters.

A retroviral vector may be used with its native, retroviral envelopeprotein, or it may be pseudotyped with a different envelope protein totarget certain cell types or to increase viral titer. Certainembodiments may employ envelope proteins derived from either retroviralor non-retroviral sources. Examples of envelope proteins derived fromnon-retroviral sources include, but are not limited to, the Sindbisvirus E2 glycoprotein and the vesticular stomatitis virus G-protein, asdescribed in U.S. Pat. Nos. 5,512,421 and 5,817,491.

The retroviral vector may be either replication competent or replicationdefective. Replication defective retroviruses are capable of infectingtarget cells in a single round infection, but do not produce infectiousvirus particles after that single round, and thus will not cause aspreading infection. In certain embodiments, the retroviral vector maycontain on a single plasmid comprising the gene of interest in additionto all the necessary retroviral coding sequences, such as the structuraland enzymatic coding sequences. In other embodiments, the retroviralvector may contain the gene of interest attached to the minimalpackaging sequences required for incorporation into a virion particle.For such minimal retroviral vectors, the vector may be introduced into aproducer cell at the same time as one or more viral packaging plasmidscontaining the structural and enzymatic viral proteins, such as thosedescribed in U.S. Pat. No. 6,506,604, or it may be introduced into apackaging cell line (e.g., PE501, PA317, ψ-2, ψ-AM, PAl2, T19-14X,VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, DAN; see also, e.g.,Miller, Human Gene Therapy, 1:5-14 (1990)); and U.S. Pat. No. 5,591,634,to form producer cell lines. The vector may introduced into the producercells through any means known in the art, such as, for example,transfection by electroporation, the use of liposomes, and/or calciumphosphate precipitation. The producer cell line generates infectiousretroviral vector particles that include the nucleic acid sequence(s)encoding the polypeptides or proteins of interest as described herein.Such retroviral vector particles then may be employed to transduceeukaryotic cells, either in vitro or in vivo. Eukaryotic cells that maybe transduced include, for example, embryonic stem cells, embryoniccarcinoma cells, hematopoietic stem cells, hepatocytes, fibroblasts,myoblasts, keratinocytes, endothelial cells, epithelial cells includinglung epithelial cells and intestinal epithelial cells, and otherculture-adapted cell lines.

Transduced cells may be further selected isolated for positive andstable expression of the polypeptides or proteins of interest, or theymay be used immediately as transiently expressing cells. Stableexpressing cells may be selected or isolated according to knowntechniques in the art, such as by flow cytometric cell sorting or bydrug selection. Stable cells may be isolated for either clonal expansionor bulk expansion of positive cells.

When transient expression of the cytoplasmic indicator protein, forexample, is desired or when cells that stably express the cytoplasmicindicator protein are difficult to obtain, epithelial cells comprisingat least one CaCC may be transfected, transformed, or transduced with arecombinant expression vector (including a plasmid or viral vector(e.g., a retroviral vector, including a lentiviral vector) prior toperforming a method to determine anion influx or to identify an agentthat inhibits a CaCC. For example, epithelial cells (e.g., a pluralityof intestinal epithelial cells or lung epithelial cells) may be infected(i.e., transduced) with a retroviral vector such as a lentiviral vectorthat comprises a polynucleotide encoding a cytoplasmic indicator proteinthat is capable of binding the halide or nitrate, under conditions andfor a time sufficient for the vector to infect the plurality ofepithelial cells. The epithelial cells are then cultured underconditions and for a time sufficient that permit expression of theindicator protein in the cytoplasm of the epithelial cell. Theepithelial cells expressing the cytoplasmic indicator protein may thenbe used in the methods as described herein for identifying a CaCCinhibitor or for determining anion influx through a CaCC.

Agents

Agents (which may also be referred to as bioactive agents) may beprovided as “libraries” or collections of compounds, compositions, ormolecules. For example, agents include low molecular weight, organicmolecules, which typically include compounds known in the art as “smallmolecules.” A small molecule may have a molecular weight less than 10⁵daltons, less than 10⁴ daltons, or less than 10³ daltons. Other agentsthat may be useful for inhibiting or blocking at least one CaCC, thusinhibiting, blocking, or reducing chloride conductance through the CaCCand inhibiting chloride secretion from a cell through a CaCC includepeptides. Peptides and small molecules may be synthesized according tomethods routinely practiced by person skilled in the synthesis ofpeptides or small molecules, respectively.

The methods described herein are useful for screening large numbers ofagents (e.g., multiple hundreds of agents) quickly. Candidate agents,such as small molecules and peptides, may be obtained from combinatoriallibraries. Combinatorial libraries of agents can be purchased from acommercial vendor or can be prepared according to methods with which askilled artisan is familiar. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example in DeWitt etal., Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993); Erb et al., Proc.Natl. Acad. Sci. USA 91:11422 (1994); Zuckermann et al., J. Med. Chem.37:2678 (1994); Cho et al., Science 261:1303 (1993); Carrell et al.,Angew. Chem. Int Ed. Engl. 33:2059 (1994); Carell et al., Angew. Chem.Int. Ed Engl. 33:2061 (1994); and in Gallop et al., J. Med. Chem.37:1233 (1994).

Candidate agents that are provided as members of a combinatoriallibrary, include synthetic agents prepared according to a plurality ofpredetermined chemical reactions performed in a plurality of reactionvessels. For example, various starting compounds may be preparedaccording to one or more of solid-phase synthesis, recorded random mixmethodologies, and recorded reaction split techniques that permit agiven constituent to traceably undergo a plurality of permutationsand/or combinations of reaction conditions. The resulting productscomprise a library that can be screened followed by iterative selectionand synthesis procedures. Such synthetic combinatorial libraries includea library of peptides (see, e.g., International Patent Application Nos.PCT/US91/08694 and PCT/US91/04666) or other compositions that mayinclude small molecules as provided herein (see, e.g., InternationalPatent Application No. PCT/US94/08542, EP Patent No. 0774464, U.S. Pat.No. 5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629, whichare hereby incorporated by reference in their entireties).

Methods for characterizing an agent, including the compounds describedherein, such as determining an effective concentration (i.e., aconcentration of agent that inhibits activation of a CaCC; inhibitsconductance of chloride through a CaCC and/or that inhibits fluidsecretion from a cell (i.e., that inhibits efflux of chloride andwater); and/or that is a therapeutically effective amount for treating acondition, disease or disorder described herein) may be performed usingtechniques and procedures described herein and routinely practiced by aperson skilled in the art. The capability of an agent to inhibit a CaCC(i.e., inhibit activation of a CaCC or inhibit halide (e.g., chloride oriodide) conductance or current) using the methods described hereininclude the use of cells, such as epithelial cells, that comprise acytoplasmic indicator protein. Other methods and techniques practiced inthe art may be used that determine whether the agent inhibits CaCC(s).Certain epithelial cells or cell lines that comprise at least one CaCC(e.g., intestinal epithelial cells, T84 and Caco-2), may not exhibitoptimum properties and characteristics for use in the methods describedherein for identifying an agent or determining anion influx. Forexample, such cells may not be readily transformed, transfected, ortransduced with a recombinant expression vector that expresses thecytoplasmic indicator protein, or if the vector can be introduced, thesignal (in the instance in which YFP is the indicator protein, thefluorescent signal) may be too weak. Such cells may not exhibit therequisite growth characteristics (i.e., propagate too slowly or fail toreach an adequate level of confluency), and/or may not sufficientlyadhere to a solid surface, particularly a plastic surface sufficientlyto withstand multiple washes. These cells expressing at least one CaCCmay still be used in other methods practiced in the art and describedherein to determine the level of chloride conductance or secretion inthe cell (see, e.g., Examples 3 and 4 herein; Hartzell et al., supra;Kidd et al., supra). In addition to use of calcium-elevating agonistsdescribed herein that bind to a cognate receptor or ligand on anepithelial cell (e.g., histamine, calcimycin, ATP, carbachol, andforskolin), a calcium-elevating agent such as thapsigargin, whichproduces calcium elevation without ligand-receptor binding orphosphoinositide signaling may also be used in assays and techniques forcharactering an agent.

Other exemplary methods include short circuit apical chloride ioncurrent measurements and patch-clamp analysis (see, e.g., Muanprasat etal., J. Gen. Physiol. 124:125-37 (2004); Ma et al., J. Clin. Invest.110:1651-58 (2002); see also, e.g., Carmeliet, Verh. K Acad. Geneeskd.Belg. 55:5-26 (1993); Hamill et al., Pflugers Arch. 391:85-100 (1981)).In patch clamp analysis, for example, ionomycin may be used to elevatethe intracellular calcium level. The agents may also be analyzed inanimal models, for example, a closed intestinal loop model of cholera,suckling mouse model of cholera, and in vivo imaging of gastrointestinaltransit (see, e.g., Takeda et al., Infect. Immun. 19:752-54 (1978)).

Aminothiophene Compounds and Aminothiazole Compounds and RelatedCompositions

Agents identified by the methods described herein include agents thatare capable of inhibiting chloride conductance through a CaCC, and thusinhibiting chloride efflux, and consequently water secretion from thecell. Provided herein are compounds that are potent CaCC inhibitors,which were identified using the methods described herein. The exemplarycompounds belong to two chemical classes, aminothiophenes (referred toherein also as Class A compounds) and aminothiazoles (referred to hereinalso as Class B compounds). These compounds, in addition to exhibitingcapability to inhibit CaCCs, exhibit high potency, favorable watersolubility, drug-like properties, and chemical stability.

Aminothiophene Compounds

In one embodiment, compounds, and compositions comprising thesecompounds, of the aminothiophene class are provided. In one embodiment,the composition comprises a physiologically acceptable excipient and acompound having the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein R¹ is hydrogen or optionally substituted alkyl; R² is hydroxy,optionally substituted alkoxy, or optionally substituted phenylamino; R³is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted cycloalkyl, optionally substituted phenyl, oroptionally substituted heterocyclyl; and n is 0, 1, or 2, and whereinthe compound of structure I comprises at least one —COOH.

In another embodiment, the composition comprises a physiologicallyacceptable excipient wherein n is 1 or 2 and the compound has thefollowing structure (IA):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,

wherein

R¹ is hydrogen or optionally substituted alkyl;

R² is hydroxy, optionally substituted alkoxy, or optionally substitutedphenylamino;

R³ is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted cycloalkyl, optionally substituted phenyl, oroptionally substituted heterocyclyl, and wherein the compound ofstructure (IA) comprises at least one —COOH.

The compound of structure I and I(A) that has at least one —COOH (i.e.,carboxy), in one embodiment, includes, but is not limited to, a compoundwherein R² is hydroxy. In another embodiment, the compound may includeR³ that is —(CH₂)₂C(═O)OH, —CH═CHC(═O)OH, cyclohexyl substituted with—COOH, or phenyl substituted with —COOH.

In a specific embodiment of structure I and I(A), R¹ is hydrogen,tert-butyl, or tert-pentyl. In yet another specific embodiment, R¹ istert-butyl or tert-pentyl. In certain embodiments, R² is —OR⁴ wherein R⁴is hydrogen or optionally substituted C₁₋₆ alkyl; or phenylaminooptionally substituted with C₁₋₆ alkoxy or C₁₋₆ alkyl. In certainembodiments, R² is —OR⁴ wherein R⁴ is hydrogen or optionally substitutedwith methyl or ethyl. In other certain embodiments, R² is phenylaminooptionally substituted with methoxy or methyl. In other specificembodiments, R³ is optionally substituted furanyl; optionallysubstituted C₁-C₆ alkyl; optionally substituted C₁-C₆ alkenyl;optionally substituted cyclohexyl; phenyl; or phenyl substituted withhalo, C₁₋₆ alkyl, C₁₋₆ alkoxy, or —COOH. In yet another specificembodiment, R³ is —(CH₂)₂C(═O)OH or —CH═CHC(═O)OH. In another specificembodiment, R³ is phenyl substituted with chloro, methyl, or methoxy. Instill another specific embodiment, R³ is cyclohexyl substituted with—COOH.

In a specific embodiment, the composition comprises the compound ofstructure (I) or (IA) wherein n is 1 and R¹ is hydrogen, tert-butyl, ortert-pentyl, and the compound has one of the following structures (Ia),(Ib), or (Ic):

In a specific embodiment of structures (Ia), (Ib), or (Ic), R² is —OR⁴wherein R⁴ is hydrogen or optionally substituted C₁₋₄ alkyl; orphenylamino optionally substituted with C₁₋₄ alkoxy or C₁₋₄ alkyl. Inanother specific embodiment, R² is —OR⁴ wherein R⁴ is hydrogen oroptionally substituted with methyl or ethyl; or phenylamino optionallysubstituted with methoxy or methyl. In another certain embodiments, R³is optionally substituted furanyl; optionally substituted C₁-C₆ alkyl;optionally substituted C₁-C₆ alkenyl; optionally substituted cyclohexyl;phenyl; or phenyl substituted with halo, C₁₋₆ alkyl, C₁₋₆ alkoxy, or—COOH. In still another embodiment, R³ is —(CH₂)₂C(═O)OH or—CH═CHC(═O)OH. In another specific embodiment, R³ is phenyl substitutedwith chloro, methyl, or methoxy. In yet another specific embodiment, R³is cyclohexyl substituted with —COOH.

In another specific embodiment, the composition described abovecomprises the compound of structure (I) or (IA) wherein n is 1 and R² is—OR⁴ wherein R⁴ is hydrogen or optionally substituted C₁₋₆ alkyl, andthe compound has the following structure (Id):

In certain specific embodiments, R⁴ is hydrogen, methyl, or ethyl. Inother specific embodiments, R¹ is hydrogen, tert-butyl, or tert-pentyl.In other certain embodiments, R¹ is tert-butyl, or tert-pentyl. In yetanother specific embodiment, R³ is optionally substituted furanyl;optionally substituted C₁-C₆ alkyl; optionally substituted C₁-C₆alkenyl; optionally substituted cyclohexyl; phenyl; or phenylsubstituted with halo, C₁₋₆ alkyl, C₁₋₆ alkoxy, or —COOH. In stillanother embodiment, R³ is —(CH₂)₂C(═O)OH or —CH═CHC(═O)OH. In anotherspecific embodiment, R³ is phenyl substituted with chloro, methyl, ormethoxy. In yet another specific embodiment, R³ is cyclohexylsubstituted with —COOH.

In another specific embodiment of the compositions described herein,wherein n is 1, the compound of structure (I) and (IA) has any one ofthe following structures (Ie), (If), (Ig), or (Ih):

wherein Y is optionally substituted lower alkyl; R⁵ is hydrogen,optionally substituted C₁₋₆ alkylene, or optionally substituted C₁₋₆alkenylene; and R⁶ is hydrogen, optionally substituted alkyl, optionallysubstituted alkoxy, —COOH, or halo. In a certain embodiment, R⁶ ishydrogen, halo, C₁₋₆ alkyl, C₁₋₆ alkoxy, or —COOH. In another certainembodiment, R⁶ is hydrogen, chloro, methyl, or methoxy. In still anotherspecific embodiment, R¹ is hydrogen, tert-butyl, or tent-pentyl and R²is —OR⁴ wherein R⁴ is hydrogen or optionally substituted C₁₋₆ alkyl. Inanother specific embodiment, R⁴ is hydrogen, methyl, or ethyl. Inanother specific embodiment, R² is phenylamino optionally substitutedwith methoxy or methyl.

In yet another specific embodiment, the composition comprises thecompound of structure (I) or (IA) wherein n is 2, R¹ is hydrogen, R² is—OR⁴, and R³ is optionally substituted phenyl, and the compound has thefollowing structure (Ii):

wherein R⁴ is hydrogen or optionally substituted C₁₋₄ alkyl and R⁶ ishydrogen, optionally substituted alkyl, optionally substituted alkoxy,or halo.

In a specific embodiment, R⁴ is hydrogen, methyl, or ethyl. In anotherspecific embodiment, R⁶ is hydrogen, chloro, optionally substituted C₁₋₆alkyl, or optionally substituted C₁₋₆ alkoxy. In yet another specificembodiment, R⁶ is hydrogen, chloro, methyl, or methoxy.

In particular specific embodiments, the compositions comprise thespecific aminothiophene compounds having a structure (I), including6-tert-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid;6-tert-butyl-2-(2-methylbenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid;6-tert-butyl-2-(3-chlorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid;2-benzamido-6-tert-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid;6-tert-butyl-2-(2-chlorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid;4-(6-tert-butyl-3-(ethoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobutanoicacid;(E)-4-(6-tert-butyl-3-(ethoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobut-2-enoicacid;2-(6-tert-butyl-3-(ethoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylcarbamoyl)cyclohexanecarboxylicacid;5-(6-tert-butyl-3-(methoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-5-oxopentanoicacid;2-(3-(ethoxycarbonyl)-6-tert-pentyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylcarbamoyl)cyclohexanecarboxylicacid;4-(6-tert-butyl-3-(methoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobutanoicacid;2-(4-methylbenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylicacid;2-benzamido-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylicacid;2-(2-chlorobenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylicacid;2-(3-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylicacid;4-(6-tert-butyl-3-(m-tolylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobutanoicacid;2-(3-methylbenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid; or4-(6-tert-butyl-3-(4-methoxyphenylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobutanoicacid. In a specific embodiment, the compound is6-tert-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid. The chemical structures of these compounds (also referred toherein as Class A compounds) are presented in Table 1.

TABLE 1 Aminothiophene Compounds Compound Structure Chemical NameCaCC_(inh)-A01

6-tert-butyl-2-(furan-2- carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene- 3-carboxylic acid CaCC_(inh)-A02

6-tert-buyl-2-(2- methylbenzamido)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylic acid CaCC_(inh)-A03

6-tert-butyl-2-(3- chlorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene- 3-carboxylic acid CaCC_(inh)-A04

2-benzamido-6-tert-butyl- 4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylic acid CaCC_(inh)-A05

6-tert-butyl-2-(2- chlorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene- 3-carboxylic acid CaCC_(inh)-A06

4-(6-tert-butyl-3- (ethoxycarbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-ylamino)-4-oxobutanoic acid CaCC_(inh)-A07

(E)-4-(6-tert-butyl-3- (ethoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen- 2-ylamino)-4-oxobut-2-enoic acidCaCC_(inh)-A08

2-(6-tert-butyl-3- (ethoxycarbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2- ylcarbamoyl)cyclohexane- carboxylic acid CaCC_(inh)-A09

5-(6-tert-butyl-3- (methoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen- 2-ylamino)-5-oxopentanoic acidCaCC_(inh)-A10

2-(3-(ethoycarbonyl)-6-tert- pentyl-4,5,6,7- tetrahydrobenzo[b]thiophen-2- ylcarbamoyl)cyclohexane- carboxylic acid CaCC_(inh)-A11

4-(6-tert-butyl-3- (methoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen- 2-ylamino)-4-oxobutanoic acid CaCC_(inh)-A12

2-(4-methylbenzamido)- 5,6,7,8-tetrahydro-4H- cyclohepta[b]thiophene-3-carboxylic acid CaCC_(inh)-A13

2-benzamido-5,6,7,8- tetrahydro-4H- cyclohepta[b]thiophene-3- carboxylicacid CaCC_(inh)-A14

2-(2-chlorobenzamido)- 5,6,7,8-tetrahydro-4H- cyclohepta[b]thiophene-3-carboxylic acid CaCC_(inh)-A15

2-(3-methoxybenzamido)- 5,6,7,8-tetrahydro-4H- cyclohepta[b]thiophene-3-carboxylic acid CaCC_(inh)-A16

4-(6-tert-butyl-3-(m- tolylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen- 2-ylamino)-4-oxobutanoic acid CaCC_(inh)-A17

2-(3-methylbenzamido)- 4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylic acid CaCC_(inh)-A18

4-(6-tert-butyl-3-(4- methoxyphenylcarbamoyl)- 4,5,6,7-tetrahydrobenzo[b]thiophen- 2-ylamino)-4-oxobutanoic acid

Aminothiazole Compounds

In one embodiment, compounds, and compositions comprising thesecompounds, of the aminothiazole class are provided. In one embodiment,the composition comprises a physiologically acceptable excipient and acompound having the following structure (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,

wherein R⁷ is optionally substituted C₁₋₆ alkyl, optionally substitutedphenyl, or optionally substituted phenylacyl;

R⁸ is hydrogen or optionally substituted C₁₋₆ alkyl or optionallysubstituted phenyl;

R⁹ and R¹⁰ are the same or different and independently hydrogen,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted phenoxy. In certain embodiments, at least one ofR⁹ and R¹⁰ is not hydrogen.

In a particular embodiment, R⁷ is methyl, ethyl, unsubstitutedphenylacyl, phenyl, or phenyl substituted with carboxy, C₁₋₆ alkyl,halo, optionally substituted cycloalkyl, or C₁₋₆ alkoxy. In otherspecific embodiments, R⁷ is phenyl substituted with C₁₋₄ alkyl, or C₁₋₄alkoxy. In yet another specific embodiment, R⁷ is phenyl substitutedwith hydroxy, chloro, bromo, methyl, ethyl, trifluoromethyl, methoxy,ethoxy, or cyclohexyl. In still another specific embodiment, R⁷ isphenyl substituted with carboxy and hydroxyl; di-halo; or C₁₋₆ alkyl andhalo. In yet another specific embodiment, R⁷ is phenyl substituted withdi-chloro or with methyl and chloro.

In another specific embodiment, R⁸ is hydrogen or optionally substitutedC₁₋₄ alkyl. In a specific embodiment, R⁸ is hydrogen, n-propyl,—CH₂C(═O)OH, or —(CH₂)₂C(═O)OH.

In a particular embodiment, R⁹ is hydrogen, optionally substituted C₁₋₄alkyl, or optionally substituted C₁₋₄ alkoxy, or phenoxy; and R¹⁰ isoptionally substituted C₁₋₄ alkyl, optionally substituted C₁₋₄ alkoxy,or phenoxy. In another particular embodiment, R⁹ is hydrogen, C₁₋₄alkyl, trifluoromethyl, methoxy, ethoxy, or phenoxy; and R¹⁰ is C₁₋₄alkyl, trifluoromethyl, methoxy, ethoxy, or phenoxy. In a specificembodiment, R⁹ is hydrogen and R¹⁰ is methyl, ethyl, isobutyl, methoxy,phenoxy, or trifluoromethyl. In another specific embodiment, R⁹ ishydrogen and R¹⁰ is methyl, ethyl, isobutyl, phenoxy, or methoxy, andR¹⁰ is located at the 4-position. In yet another specific embodiment, R⁹is hydrogen and R¹⁰ is trifluoromethyl and R¹⁰ is located at the3-position. In still another specific embodiment, R⁹ is methyl andlocated at the 2-position, and R¹⁰ is methyl and located at the4-position. In certain embodiments, the compound of substructure IIacomprises at least one —COOH.

In another embodiment, the composition comprises the compound ofstructure (II) wherein R⁷ is optionally substituted phenyl and thecompound has the following substructure (IIa):

wherein R¹¹ and R¹² are the same or different and independentlyhydrogen, hydroxy, carboxy, halo, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl.

In one specific embodiment of the substructure (IIa), R⁹ and R¹⁰ are thesame or different and independently hydrogen, optionally substitutedC₁₋₄ alkyl, or optionally substituted C₁₋₄ alkoxy, or phenoxy. Inanother specific embodiment, R⁹ and R¹⁰ are the same or different andindependently hydrogen, C₁₋₄ alkyl, trifluoromethyl, methoxy, ethoxy, orphenoxy. In still another specific embodiment R⁹ is hydrogen and R¹⁰ ismethyl, ethyl, isobutyl, methoxy, phenoxy, or trifluoromethyl. In yetanother specific embodiment, R⁹ is hydrogen and R¹⁰ is methyl, ethyl,isobutyl, phenoxy, or methoxy, and wherein R¹⁰ is located at the4-position. In still another specific embodiment, R⁹ is hydrogen and R¹⁰is trifluoromethyl and R¹⁰ is located at the 3-position. In anotherspecific embodiment, R⁹ is methyl and located at the 2-position, andwherein R¹⁰ is methyl and located at the 4-position. In other specificembodiments of the substructure (IIa), R⁸ is hydrogen or optionallysubstituted C₁₋₆ alkyl. In another specific embodiment, R⁸ is hydrogen,n-propyl, —CH₂C(═O)OH, or —(CH₂)₂C(═O)OH. In specific embodiments of thesubstructure (IIa), R¹¹ and R¹² are the same or different andindependently hydrogen, hydroxy, carboxy, halo, optionally substitutedC₁₋₄ alkyl, C₁₋₄ alkoxy, or optionally substituted cyclohexyl. In stillother embodiments, R¹¹ is hydrogen and R¹² is hydrogen, hydroxy,carboxy, bromo, chloro, trifluoromethyl, methyl, ethyl, isobutyl,methoxy, ethoxy, or cyclohexyl. In certain specific embodiment, R¹¹ ishydroxy and R¹² is carboxy. In other certain specific embodiments, R¹¹is methyl and R¹² is halo. In still another specific embodiment, R¹¹ ishydrogen and R¹² is chloro or wherein each of R¹¹ and R¹² is chloro. Incertain embodiments, the compound of substructure IIa comprises at leastone —COOH.

In another embodiment, the composition comprises the compound ofstructure (II) R⁸ is hydrogen or Y and the compound has the followingstructures (IIb) or (IIc):

wherein Y is optionally substituted C₁₋₄ alkyl.

In specific embodiments of the substructures (IIb) and (IIc), Y ismethyl, ethyl, n-propyl, —CH₂C(═O)OH, or —(CH₂)₂C(═O)OH.

In another specific embodiment of the substructures (IIb) and (IIc), R⁷is methyl, ethyl, unsubstituted phenylacyl, phenyl, or phenylsubstituted with carboxy, C₁₋₆ alkyl, halo, optionally substitutedcycloalkyl, or C₁₋₆ alkoxy. In yet another specific embodiment, R⁷ isphenyl substituted with hydroxy, chloro, bromo, methyl, ethyl,trifluoromethyl, methoxy, ethoxy, or cyclohexyl. In still anotherspecific embodiment, R⁷ is phenyl substituted with carboxy and hydroxyl;di-halo; or C₁₋₆ alkyl and halo. In yet another specific embodiment, R⁷is phenyl substituted with di-chloro or with methyl and chloro.

In other specific embodiments of the substructures (IIb) and (IIc), R⁹and R¹⁰ are the same or different and independently hydrogen, optionallysubstituted C₁₋₄ alkyl, or optionally substituted C₁₋₄ alkoxy, orphenoxy. In another specific embodiment, R⁹ and R¹⁰ are the same ordifferent and independently hydrogen, C₁₋₄ alkyl, trifluoromethyl,methoxy, ethoxy, or phenoxy. In still another specific embodiment R⁹ ishydrogen and R¹⁰ is methyl, ethyl, isobutyl, methoxy, phenoxy, ortrifluoromethyl. In yet another specific embodiment, R⁹ is hydrogen andR¹⁰ is methyl, ethyl, isobutyl, phenoxy, or methoxy, and wherein R¹⁰ islocated at the 4-position. In still another specific embodiment, R⁹ ishydrogen and R¹⁰ is trifluoromethyl and R¹⁰ is located at the3-position. In another specific embodiment, R⁹ is methyl and located atthe 2-position, and wherein R¹⁰ is methyl and located at the 4-position.

In another embodiment, the composition comprises the compound ofstructure (II) wherein R⁹ is hydrogen and R¹⁰ is methyl,trifluoromethyl, or —OR¹⁴; or each of R⁹ and R¹⁰ is methyl, and thecompound has the following substructure (IIe), (IIf), (IIg), or (IIh):

wherein R¹⁴ is hydrogen, optionally substituted C₁₋₆ alkyl, oroptionally substituted phenyl.

In specific embodiments of the substructures (IId), (IIe), (IIf), and(IIh), R¹⁴ is hydrogen, methyl, or unsubstituted phenyl. In otherspecific embodiments, R⁷ is methyl, ethyl, unsubstituted phenylacyl,phenyl, or phenyl substituted with carboxy, C₁₋₆ alkyl, halo, optionallysubstituted cycloalkyl, or C₁₋₆ alkoxy. In yet another specificembodiment, R⁷ is substituted phenyl, wherein phenyl is substituted withhydroxy, chloro, bromo, methyl, ethyl, trifluoromethyl, methoxy, ethoxy,or cyclohexyl. In still another specific embodiment, R⁷ is phenylsubstituted with carboxy and hydroxyl; di-halo; or C₁₋₄ alkyl and halo.In yet another specific embodiment, R⁷ is phenyl substituted either withdi-chloro or with methyl and chloro. In yet other specific embodimentsof the substructures (IId), (11e), (I11), and (IIh), R⁸ is hydrogen oroptionally substituted C₁₋₆ alkyl. In another specific embodiment, R⁸ ishydrogen, n-propyl, —CH₂C(═O)OH, or —(CH₂)₂C(═O)OH.

In particular specific embodiments, the compositions comprise thespecific Aminothiazole compounds having a structure (II), including2-hydroxy-5-(4-p-tolylthiazol-2-ylamino)benzoic acid;2-(2-(3-chloro-4-methylphenylamino)-4-p-tolylthiazol-5-yl)acetic acid;2-(2-(3-bromophenylamino)-4-p-tolylthiazol-5-yl)acetic acid;2-(2-(2,4-dichlorophenylamino)-4-p-tolylthiazol-5-yl)acetic acid;2-(4-p-tolyl-2-(3-(trifluoromethyl)phenylamino)thiazol-5-yl)acetic acid;4-(4-(2,4-dimethylphenyl)-5-propylthiazol-2-ylamino)benzoic acid;2-(2-(4-bromophenylamino)-4-(2,4-dimethylphenyl)thiazol-5-yl)aceticacid;2-(4-(2,4-dimethylphenyl)-2-(4-(trifluoromethyl)phenylamino)thiazol-5-yl)aceticacid; N-(4-isobutylphenyl)-4-(4-phenoxyphenyl)thiazol-2-amine;N-(4-cyclohexylphenyl)-4-(3-(trifluoromethyl)phenyl)thiazol-2-amine;2-(2-(4-ethoxyphenylamino)-4-p-tolylthiazol-5-yl)acetic acid;3-methyl-N-(5-phenyl-4-p-tolylthiazol-2-yl)benzamide;N-ethyl-4-p-tolylthiazol-2-amine; or3-(4-(4-methoxyphenyl)-2-(4-methoxyphenylamino)thiazol-5-yl)propanoicacid. In a specific embodiment, the compound is2-hydroxy-5-(4-p-tolylthiazol-2-ylamino)benzoic acid. The chemicalstructures corresponding to these aminothiazole compounds of structure(II) (also referred to herein as Class B compounds) are presented inTable 2.

TABLE 2 Aminothiazole Compounds Compound Structure Chemical NameCaCC_(inh)-B01

2-hydroxy-5-(4-p- tolylthiazol-2- ylamino)benzoic acid CaCC_(inh)-B02

2-(2-(3-chloro-4- methylphenylamino)-4-p- tolylthiazol-5-yl)acetic acidCaCC_(inh)-B03

2-(2-(3-bromophenylamino)- 4-p-tolylthiazol-5-yl)acetic acidCaCC_(inh)-B04

2-(2-(2,4- dichlorophenylamino)-4-p- tolylthiazol-5-yl)acetic acidCaCC_(inh)-B05

2-(4-p-tolyl-2-(3- (trifluoromethyl)phenylamino) thiazol-5-yl)aceticacid CaCC_(inh)-B06

4-(4-(2,4-dimethylphenyl)-5- propylthiazol-2- ylamino)benzoic acidCaCC_(inh)-B07

2-(2-(4-bromophenylamino)- 4-(2,4- dimethylphenyl)thiazol-5- yl)aceticacid CaCC_(inh)-B08

2-(4-(2,4-dimethylphenyl)-2- (3- (trifluoromethyl)phenylamino)thiazol-5-yl)acetic acid CaCC_(inh)-B09

N-(4-isobutylphenyl)-4-(4- phenoxyphenyl)thiazol-2- amine CaCC_(inh)-B10

N-(4-cyclohexylphenyl)-4- (3- (trifluoromethyl)phenyl) thiazol-2-amineCaCC_(inh)-B11

2-(2-(4-ethoxyphenylamino)- 4-p-tolylthiazol-5-yl)acetic acidCaCC_(inh)-B12

3-methyl-N-(5-phenyl-4-p- tolylthiazol-2-yl)benzamide CaCC_(inh)-B13

N-ethyl-4-p-tolylthiazol-2- amine CaCC_(inh)-B14

3-(4-(4-methoxyphenyl)-2- (4- methoxyphenylamino)thiazol- 5-yl)propanoicacid

The agents, including the aminothiophene compounds having the structureof formula I and substructures of formula IA, Ia-Ii and aminothiazolecompounds having the structure of formula II and substructures offormula IIa-IIh as described herein, are capable of inhibiting orblocking a CaCC channel or pore (blocking or impeding activation of aCaCC channel or pore) located in the outer cell membrane of a cell andthus inhibiting CaCC chloride conductance. Also provided herein aremethods of inhibiting a calcium-activated chloride channel comprisingcontacting a cell (e.g., as an epithelial cell, including an intestinalepithelial cell and a lung epithelial cell) that comprises at least onecalcium-activated chloride channel with a compound (or compositioncomprising the compound) (e.g., a aminothiophene compound having thestructure of formula I and substructures of formula I(A), Ia-Ii or anaminothiazole compound having the structure of formula II andsubstructures of formula IIa-IIh), under conditions and for a timesufficient for the cell and the compound to interact, and in an amounteffective to inhibit activation of the channel.

The aminothiophene compounds having the structure of formula I andsubstructures of formula I(A) and Ia-Ii or an aminothiazole compoundhaving the structure of formula II and substructures of formula IIa-IIhand compositions comprising these compounds are useful for treating acondition, disease or disorder in a subject that is characterized by,caused by, or exacerbated by aberrantly increased calcium-activatedchloride channel activity. As described in greater detail herein, suchdiseases and disorders include diseases and disorders related to (orassociated with) excess intestinal secretion of fluids, such assecretory diarrhea, and diseases and disorders associated with excessmucus production, such as cystic fibrosis, asthma, chronic obstructivepulmonary disease, and bronchiectasis. The compounds and compositionscomprising the compounds may be used to inhibit (i.e., decrease orreduce) fluid secretion from a cell, particularly a cell from whichfluid secretion is abnormally increased, which in part, may result fromaberrantly increased chloride conductance through a CaCC. Thesecompounds and compositions may be contacted with the cell (oradministered to a subject in need thereof) in an amount effective toinhibit (decrease or reduce) chloride conductance through at least onecalcium-activated chloride channel, which thereby results in decreasedchloride secretion and decreased fluid secretion from the cell. Incertain embodiments of the methods described herein, the cell, whichcomprises at least one calcium-activated chloride channel, may be anepithelial cell, which in certain embodiments is an intestinalepithelial cell or a pulmonary epithelial cell.

Chemistry Definitions

Certain chemical groups named herein are preceded by a shorthandnotation indicating the total number of carbon atoms that are to befound in the indicated chemical group. For example, C₁-C₆ alkyldescribes an alkyl group, as defined below, having a total of 1 to 6carbon atoms, and C₅-C₇ cycloalkyl describes a cycloalkyl group, asdefined below, having a total of 5 to 7 carbon atoms. The total numberof carbons in the shorthand notation does not include carbons that mayexist in substituents of the group described. In addition to theforegoing, as used herein, unless specified to the contrary, thefollowing terms have the meaning indicated.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 10carbon atoms, while the terms “C₁₋₄ alkyl” and “C₁₋₆ alkyl” have thesame meaning as alkyl but contain from 1 to 4 carbon atoms and 1 to 6carbon atoms, respectively. A lower alkyl refers to an alkyl that hasany number of carbon atoms between 1 and 6. Representative saturatedstraight chain alkyls include methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, and the like, while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, tent-pentyl, heptyl,n-octyl, isopentyl, 2-ethylhexyl and the like. Representative saturatedcyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—CH₂cyclopropyl, —CH₂cyclobutyl, —CH₂cyclopentyl, —CH₂cyclohexyl, andthe like; unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Cyclic alkyls, also referred to as“homocyclic rings,” include di- and poly-homocyclic rings such asdecalin and adamantyl.

Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,”respectively). “Alkylene” or “alkylene chain” refers to a straight orbranched divalent hydrocarbon chain linking the rest of the molecule toa radical group, consisting solely of carbon and hydrogen, containing nounsaturation and having from one to twelve carbon atoms, for example,methylene, ethylene, propylene, n-butylene, and the like. The alkylenechain is attached to the rest of the molecule through a single bond andto the radical group through a single bond. The points of attachment ofthe alkylene chain to the rest of the molecule and to the radical groupcan be through one carbon in the alkylene chain or through any twocarbons within the chain. Unless stated otherwise specifically in thespecification, an alkylene chain may be optionally substituted by one ormore of the following substituents: halo, cyano, nitro, aryl,cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl,—OR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(p)R^(a) (where p is 0, 1 or 2), and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocylylalkyl, heteroaryl or heteroarylalkyl.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onedouble bond and having from two to twelve carbon atoms, for example,ethenylene, propenylene, n-butenylene, and the like. The alkenylenechain is attached to the rest of the molecule through a double bond or asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkenylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, an alkenylene chain may be optionally substituted byone or more of the following substituents: halo, cyano, nitro, aryl,cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl,—OR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(p)R^(a) (where p is 0, 1 or 2), and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl,haloalkyl, cycloalkyl, cycloalkylalkyl, aryl (optionally substitutedwith one or more halo groups), aralkyl, heterocyclyl, heterocylylalkyl,heteroaryl or heteroarylalkyl, and where each of the above substituentsis unsubstituted unless otherwise indicated.

Representative straight chain and branched alkenyls include ethylenyl,propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; representative straight chain and branched alkynyls includeacetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1 butynyl, and the like.

It is understood that within the context of the compounds describedherein that the terms alkyl, aryl, arylalkyl, heterocycle, homocycle,and heterocycloalkyl are taken to comprise unsubstituted alkyl andsubstituted alkyl, unsubstituted aryl and substituted aryl,unsubstituted arylalkyl and substituted arylalkyl, unsubstitutedheterocycle and substituted heterocycle, unsubstituted homocycle andsubstituted homocycle, unsubstituted heterocycloalkyl and substitutedheterocyclealkyl, respectively, as defined herein, unless otherwisespecified.

As used herein, the term “substituted” in the context of alkyl, alkoxy,aryl, arylalkyl, cycloalkyl, heterocycle, and heterocycloalkyl meansthat at least one hydrogen atom of the alky, aryl, arylalkyl,heterocycle, or heterocycloalkyl moiety is replaced with a substituent.The term “optionally substituted” as used in the context of anoptionally substituted alkyl, alkoxy, aryl, arylalkyl, cycloalkyl,heterocycle, and heterocycloalkyl means that when the alkyl, alkoxy,aryl, arylalkyl, cycloalkyl, heterocycle, and heterocycloalkyl,respectively, is substituted, t least one hydrogen atom of the alky,aryl, arylalkyl, heterocycle, or heterocycloalkyl moiety is replacedwith a substituent. In the instance of an oxo substituent (“═O”) twohydrogen atoms are replaced. A “substituent” as used within the contextof this disclosure includes oxo, halogen, hydroxy, cyano, nitro, amino,alkylamino, dialkylamino, alkyl, alkoxy, thioalkyl, haloalkyl,substituted alkyl, heteroalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocycloalkyl, substituted heterocycloalkyl,—NR_(a)R_(b), —NR_(a)C(═O)R_(b), —NR_(a)C(═O)NR_(a)R_(b),—NR_(a)C(═O)OR_(b), —NR_(a)S(═O)₂R_(b), —OR_(a),—C(═O)R_(a)—C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OCH₂C(═O)NR_(a)R_(b),—OC(═O)NR_(a)R_(b), —SH, —SR_(a), —SOR_(a), —S(═O)₂NR_(a)R_(b),—S(═O)₂R_(a), —SR_(a)C(═O)NR_(a)R_(b), —OS(═O)₂R_(a) and —S(═O)₂OR_(a)(also written as —SO₃R_(a)), wherein R_(a) and R_(b) are the same ordifferent and independently hydrogen, alkyl, haloalkyl, substitutedalkyl, alkoxy, aryl, substituted aryl, arylalkyl, substituted arylalkyl,arylalkoxy, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heterocycle, substituted heterocycle,heterocycloalkyl or substituted heterocycloalkyl. The definitions ofR_(a) and R_(b) above apply to all uses of these substituents throughoutthe description.

Representative substituents include (but are not limited to) alkoxy(i.e., alkyl-O—, including C₁₋₆ alkoxy and C₁₋₄ alkoxy (e.g., methoxy,ethoxy, propoxy, butoxy)), aryloxy (e.g., phenoxy, chlorophenoxy,tolyloxy, methoxyphenoxy, benzyloxy, alkyloxycarbonylphenoxy,alkyloxycarbonyloxy, acyloxyphenoxy), acyloxy (e.g., propionyloxy,benzoyloxy, acetoxy), carbamoyloxy, carboxy, mercapto, alkylthio,acylthio, arylthio (e.g., phenylthio, chlorophenylthio, alkylphenylthio,alkoxyphenylthio, benzylthio, alkyloxycarbonyl-phenylthio), amino (e.g.,amino, mono- and di-C₁-C₃ alkanylamino, methylphenylamino,methylbenzylamino, C₁-C₃ alkanylamido, acylamino, carbamamido, ureido,guanidino, nitro and cyano). Moreover, any substituent may have from 1-5further substituents attached thereto.

“Aryl” means an aromatic carbocyclic moiety such as phenyl or naphthyl(i.e., naphthalenyl) (1- or 2-naphthyl) or anthracenyl (e.g.,2-anthracenyl).

“Arylalkyl” (e.g., phenylalkyl) means an alkyl having at least one alkylhydrogen atom replaced with an aryl moiety, such as —CH₂-phenyl,—CH═CH-phenyl, —C(CH₃)═CH-phenyl, and the like.

“Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least 1 carbon atom, including both mono- andbicyclic ring systems. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl (including 6-quinolinyl),isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, and quinazolinyl.

“Heteroarylalkyl” means an alkyl having at least one alkyl hydrogen atomreplaced with a heteroaryl moiety, such as —CH₂pyridinyl,—CH₂pyrimidinyl, and the like.

“Heterocycle” (also referred to herein as a “heterocyclic ring”) means a4- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclicring which is either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms maybe optionally oxidized, and the nitrogen heteroatom may be optionallyquaternized, including bicyclic rings in which any of the aboveheterocycles are fused to a benzene ring. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined herein. Thus, in addition to the heteroarylslisted above, heterocycles also include morpholinyl, pyrrolidinonyl,pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

Phenylamino refers to —N(R_(a))-phenyl. The phenyl may be optionallysubstituted as described herein, for example, with C₁-C₆ alkoxy (such asmethoxy or ethyoxy) or C₁-C₆ alkyl (e.g., methyl or ethyl).

Phenylacyl refers to —C(O)-phenyl. Phenyl may be optionally substitutedwith substituents R_(a) described herein.

“Heterocycloalkyl” means an alkyl having at least one alkyl hydrogenatom replaced with a heterocycle, such as —CH₂morpholinyl,—CH₂CH₂piperidinyl, —CH₂azepineyl, —CH₂pirazineyl, —CH₂pyranyl,—CH₂furanyl, —CH₂pyrrolidinyl, and the like.

“Homocycle” (also referred to herein as “homocyclic ring”) means asaturated or unsaturated (but not aromatic) carbocyclic ring containingfrom 3-7 carbon atoms, such as cyclopropane, cyclobutane, cyclopentane,cyclohexane, cycloheptane, cyclohexene, and the like.

“Halogen” or “halo” means fluoro, chloro, bromo, and iodo.

“Haloalkyl,” which is an example of a substituted alkyl, means an alkylhaving at least one hydrogen atom replaced with halogen, such astrifluoromethyl and the like.

“Haloaryl,” which is an example of a substituted aryl, means an arylhaving at least one hydrogen atom replaced with halogen, such as4-fluorophenyl and the like.

“Alkoxy” means an alkyl moiety attached through an oxygen bridge (i.e.,—O-alkyl) such as methoxy, ethoxy, and the like.

“COOH” means a carboxy and may also be set forth as —C(═O)OH.

“Haloalkoxy,” which is an example, of a substituted alkoxy, means analkoxy moiety having at least one hydrogen atom replaced with halogen,such as chloromethoxy and the like.

“Alkoxydiyl” means an alkyl moiety attached through two separate oxygenbridges (i.e., —O-alkyl-O—) such as —O—CH₂—O—, —O—CH₂CH₂—O—,—O—CH₂CH₂CH₂—O—, —O—CH(CH₃)CH₂CH₂—O—, —O—CH₂C(CH₃)₂CH₂—O—, and the like.

“Alkanediyl” means a divalent alkyl from which two hydrogen atoms aretaken from the same carbon atom or from different carbon atoms, such as—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂—, —CH₂C(CH₃)₂CH₂—, and thelike.

“Thioalkyl” means an alkyl moiety attached through a sulfur bridge(i.e., —S-alkyl) such as methylthio, ethylthio, and the like.

“Alkylamino” and “dialkylamino” mean one or two alkyl moieties attachedthrough a nitrogen bridge (i.e., —N-alkyl) such as methylamino,ethylamino, dimethylamino, diethylamino, and the like.

“Carbamate” is —R_(a)OC(═O)NR_(a)R_(b).

“Cyclic carbamate” means any carbamate moiety that is part of a ring.

“Amidyl” is —NR_(a)R_(b).

“Hydroxyl” or “hydroxy” refers to the —OH radical.

“Sulfhydryl” or “thio” is —SH.

“Amino” refers to the —NH₂ radical.

“Nitro” refers to the —NO₂ radical.

“Imino” refers to the ═NH radical.

“Thioxo” refers to the ═S radical.

“Cyano” refers to the —C≡N radical.

“Sulfonamide refers to the radical —S(═O)₂NH₂.

“Isocyanate” refers to the N═C═O radical.

“Isothiocyanate” refers to the —N═C═S radical.

“Azido” refers to the —N═N⁺═N⁻ radical.

“Carboxy” refers to the —CO₂H radical (also depicted as —C(═O)OH and—COOH).

“Hydrazide” refers to the —C(═O)NR_(a)—NR_(a)R_(b) radical.

“Oxo” refers to the ═O radical.

The compounds described herein may generally be used as the free acid orfree base. Alternatively, the compounds may be used in the form of acidor base addition salts. Acid addition salts of the free base aminocompounds may be prepared according to methods well known in the art,and may be formed from organic and inorganic acids. Suitable organicacids include (but are not limited to) maleic, fumaric, benzoic,ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic,tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic,aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonicacids. Suitable inorganic acids include (but are not limited to)hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Baseaddition salts of the free acid compounds of the compounds describedherein may also be prepared by methods well known in the art, and may beformed from organic and inorganic bases. Suitable inorganic basesincluded (but are not limited to) the hydroxide or other salt of sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum, and the like, and organic bases such as substitutedammonium salts. Thus, the term “pharmaceutically acceptable salt” ofstructure (I) or of structure (II), as well as any and all substructuresdescribed herein, is intended to encompass any and all pharmaceuticallysuitable salt forms.

Also contemplated are prodrugs of any of the compounds described herein.Prodrugs are any covalently bonded carriers that release a compound ofstructure (I), as well as any of the substructures herein, in vivo whensuch prodrug is administered to a subject. Prodrugs are generallyprepared by modifying functional groups in a way such that themodification is cleaved, either by routine manipulation or by an in vivoprocess, yielding the parent compound. Prodrugs include, for example,compounds described herein when, for example, hydroxy or amine groupsare bonded to any group that, when administered to a subject, is cleavedto form the hydroxy or amine groups. Thus, representative examples ofprodrugs include (but are not limited to) acetate, formate and benzoatederivatives of alcohol and amine functional groups of the compounds ofstructure (I), as well as any of the substructures herein. Further, inthe case of a carboxylic acid (—COOH), esters may be employed, such asmethyl esters, ethyl esters, and the like. Prodrug chemistry isconventional to and routinely practiced by a person having ordinaryskill in the art.

Prodrugs are typically rapidly transformed in vivo to yield the parentcompound (i.e., an aminothiophene compound having a structure I orsubstructures Ia-Ii; or an aminothiazole compound having a structure IIor substructures IIa-Ih), for example, by hydrolysis in blood. Theprodrug compound often offers advantages of solubility, tissuecompatibility or delayed release in a mammalian organism (see, e.g.,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., etal., “Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series,Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B.Roche, American Pharmaceutical Association and Pergamon Press, 1987,both of which are incorporated in full by reference herein.

With regard to stereoisomers, the compounds of structure (I) andstructure (II), as well as any substructure herein, may have one or morechiral centers and may occur in any isomeric form, including racemates,racemic mixtures, and as individual enantiomers or diastereomers. Inaddition, the compounds of structure (I) and structure (II), as well asany substructure thereof, include E and Z isomers of all double bonds.All such isomeric forms of the compounds are included and contemplated,as well as mixtures thereof. Furthermore, some of the crystalline formsof any compound described herein may exist as polymorphs, which are alsoincluded and contemplated by the present disclosure. In addition, someof the compounds may form solvates with water or other organic solvents.Such solvates are similarly included within the scope of compounds andcompositions described herein.

In general, the compounds used in the reactions described herein may bemade according to organic synthesis techniques known to those skilled inthis art, starting from commercially available chemicals and/or fromcompounds described in the chemical literature. “Commercially availablechemicals” may be obtained from standard commercial sources includingAcros Organics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis.,including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton ParkUK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada),Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), CrescentChemical Co. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman KodakCompany (Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hanover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), and Wako Chemicals USA, Inc.(Richmond Va.).

Methods known to one of ordinary skill in the art may be identifiedthrough various reference books and databases. Suitable reference booksand treatises that detail the synthesis of reactants useful in thepreparation of compounds and bioactive agents described herein, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions,” 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Additionalsuitable reference books and treatises that detail the synthesis ofreactants useful in the preparation of compounds and agents describedherein, or provide references to articles that describe the preparation,include for example, Fuhrhop, J. and Penzlin G. “Organic Synthesis:Concepts, Methods, Starting Materials”, Second, Revised and EnlargedEdition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, R. V.“Organic Chemistry, An Intermediate Text” (1996) Oxford UniversityPress, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations” 2nd Edition(1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced OrganicChemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) JohnWiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) “Modern CarbonylChemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai's1992 Guide to the Chemistry of Functional Groups” (1992) InterscienceISBN: 0-471-93022-9; Quin, L. D. et al. “A Guide to OrganophosphorusChemistry” (2000) Wiley-Interscience, ISBN: 0-471-31824-8; Solomons, T.W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN:0-471-19095-0; Stowell, J. C., “Intermediate Organic Chemistry” 2ndEdition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “IndustrialOrganic Chemicals: Starting Materials and Intermediates: An Ullmann'sEncyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over 55volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73volumes.

Specific and analogous reactants may also be identified through theindices of known chemicals prepared by the Chemical Abstract Service ofthe American Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., may be contacted for more details).Chemicals that are known but not commercially available in catalogs maybe prepared by custom chemical synthesis houses; where many of thestandard chemical supply houses (e.g., those listed above) providecustom synthesis services. A reference for the preparation and selectionof pharmaceutical salts of the compounds and bioactive agents describedherein is P. H. Stahl & C. G. Wermuth “Handbook of PharmaceuticalSalts”, Verlag Helvetica Chimica Acta, Zurich, 2002.

Synthesis of Aminothiophene and Aminothiazole Compounds

Synthesis of Aminothiophene Compounds

The compounds described herein may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples. In general, the compounds of structure (I) above may bemade by the following Reaction Scheme 1, wherein all substituents are asdefined above unless indicated otherwise.

Referring to Reaction Scheme 1, ketones of formula II and cyanoacetatesof formula 12 can be purchased or prepared according to procedures knownto those skilled in the art. Compounds of formula 11 and 12 can bereacted together in the presence of elemental sulfur and morpholine in asolvent, such as methanol, to obtain compounds of formula 13. Acidchlorides of formula 14 can be purchased or prepared according toprocedures known to those skilled in the art and reacted with compoundsof formula 3 in the presence of an appropriate base, such asdiisopropylethylamine (DIEA), in a solvent, such as dichloromethane, toobtain compounds of formula (I) and (I(A)). One skilled in the art willrecognize that appropriate protection/deprotection chemistry may berequired to obtain the desired compound of formula (I) and (I(A)).

In general, the compounds of structure (II) above may be made by thefollowing Reaction Scheme 2, wherein all substituents are as definedabove unless indicated otherwise.

Referring to Reaction Scheme 2, amines of formula 15 can be purchased orprepared according to methods known to those skilled in the art andreacted with thiophosgene (16) in the presence of water and anappropriate acid, such as hydrochloric acid, at 0° C. to obtainthioisocyanates of formula 17. Compounds of formula 17 can then betreated with aqueous ammonium hydroxide to obtain thioamides of formula18. Bromoketones of formula 19 can be purchased or prepared according tomethods known to those skilled in the art and reacted with compounds offormula 18 to obtain compounds of formula (II). One skilled in the artwill recognize that appropriate protection/deprotection chemistry may berequired to obtain the desired compound of formula (II).

Methods of Using the Compounds and Pharmaceutical Compositions

As described herein, the aminothiophene and aminothiazole compounds arecapable of inhibiting CaCC activity (i.e., inhibiting, reducing,decreasing, blocking conductance of chloride ion (i.e., chloride) in theCaCC channel or pore in a statistically significant or biologicallysignificant manner) in a cell and may be used for treating diseases,disorders, and conditions that result from, are associated with, or arerelated to aberrantly increased CaCC activity and which conditions,diseases, and disorders are thereby treatable by inhibiting CaCCactivity (including inhibiting activation of CaCC), which inhibits(decreases or reduces) chloride conductance through the CaCC andinhibits fluid secretion (or water secretion) from the cell (i.e.,efflux of chloride and water). Accordingly, methods of inhibitingchloride conductance or movement through a CaCC are provided herein thatcomprise contacting a cell (e.g., an epithelial cell including anintestinal epithelial cell and a lung epithelial cell) that comprises atleast one CaCC in the outer membrane of the cell (i.e., a cell thatexpresses a CaCC and has channels or pores formed by the CaCC in thecell membrane) with any one or more of the compounds described herein,under conditions and for a time sufficient for the CaCC and the compoundto interact. Inhibiting movement of chloride and water through the atleast one CaCC thereby inhibits (or decreases) fluid secretion from thecell. A CaCC that may be inhibited by any one or more of the compoundsor compositions described herein includes but is not limited to TMEM16A.

Accordingly, in one embodiment a method is provided for treating acondition, disease, or disorder that is associated with abnormallyincreased chloride ion conductance by administering to a subject, inneed thereof, a composition comprising at least one aminothiophenecompound (a compound having structure I or a substructure thereof)and/or at least one aminothiazole compound (a compound having structureII or a substructure thereof), or composition comprising the at leastone compound, which are described in detail herein. The compound isadministered in an amount effective to inhibit a calcium-activatedchloride channel or to inhibit activation of the CaCC, therebyinhibiting chloride ion conductance and consequently inhibiting fluidsecretion from the cell.

In certain embodiments, such as for practicing an in vitro assay methoddescribed herein, the cell may be obtained from a subject or from abiological sample. A biological sample may be a blood sample (from whichserum or plasma may be prepared and cells isolated), biopsy specimen,body fluids (e.g., lung lavage, sputum, ascites, mucosal washings,synovial fluid, peritoneal washing), bone marrow, lymph nodes, tissueexplant, organ culture, or any other tissue or cell preparation from asubject or a biological source. A biological sample may further refer toa tissue or cell preparation in which the morphological integrity orphysical state has been disrupted, for example, by dissection,dissociation, solubilization, fractionation, homogenization, biochemicalor chemical extraction, pulverization, lyophilization, sonication, orany other means for processing a biological sample derived from asubject or biological source. The subject or biological source may be ahuman or non-human animal, a primary cell culture (e.g., epithelialcells isolated from intestinal or lung tissue, or virus infected cells),or culture adapted cell line, including but not limited to, geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences, immortalized orimmortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,and the like.

Diseases and disorders that may be treated by administering acomposition comprising a compound of structure I and relatedsubstructures or a compound of structure II and any related substructuredescribed herein include aberrantly increased intestinal fluidsecretion, such as secretory diarrhea. Secretory diarrhea can resultfrom exposure to a variety of enteropathogenic organisms (i.e., entericpathogen) including, without limitation, bacteria such as cholera(Vibrio cholera), E. coli (particularly enterotoxigenic (ETEC)),Shigella, Salmonella, Campylobacter, Clostridium difficile; parasites(e.g., Giardia, Entamoeba histolytica, Cryptosporidiosis, Cyclospora);and diarrheal viruses (e.g., rotavirus, Group A and Group C; norovirus,sapovirus). Secretory diarrhea may also be a disorder or sequelaeassociated with food poisoning, or exposure to a toxin including anenterotoxin such as cholera toxin, a E. coli toxin, a Salmonella toxin,a Campylobacter toxin, or a Shigella toxin.

Other secretory diarrheas that may be treated by administering thecompounds and compositions comprising the compounds described hereininclude diarrhea associated with or that is a sequelae of AIDS, diarrheathat is a condition related to the effects of anti-AIDS medications suchas protease inhibitors, diarrhea that is a condition or is related toadministration of chemotherapeutic compounds, inflammatorygastrointestinal disorders, such as ulcerative colitis, inflammatorybowel disease (IBD), Crohn's disease, diverticulosis, and the like.Intestinal inflammation modulates the expression of three majormediators of intestinal salt transport and may contribute to diarrhea inulcerative colitis both by increasing transepithelial Cl⁻ secretion andby inhibiting the epithelial NaCl absorption (see, e.g., Lohi et al.,Am. J. Physiol. Gastrointest. Liver Physiol. 283:G567-75 (2002)).

Other diseases or disorders that may be treated by administering to asubject in need thereof, the compounds (aminothiophene compounds havingthe structure of formula I and substructures of formula I(A), Ia-Ii andaminothiazole compounds having the structure of formula II andsubstructures of formula IIa-IIh) include diseases and disordersassociated with abnormally increased mucus secretion (i.e., abnormallyincreased mucus secretion is a condition associated with or is asequelae of the disease or disorder). Accordingly, the compounds andcompositions thereof may be used for treating asthma, cystic fibrosis,bronchiectasis, and chronic pulmonary disease (see, e.g., Wang et al.,Cell Biol. Int. 31(11):1388-95 (2007), Epub 2007 June 29; Yasuo et al.,Respiration 73:347-359 (2006); Shale et al., Eur. Respir. J. 23:797-798(2004); Wang et al., Chinese Med. J. 120:1051-57 (2007); Barnes, Curr.Drug Targets Inflamm. Allergy. 4:675-83 (2005); Cuthbert, J. R. Soc.Med. 99:30-35 (2006); Hegab et al., Chest 131:1149-56 (2007)).

Methods are also provided herein for treating a disease or disorderassociated with abnormally or aberrantly increased chloride ionsecretion, wherein the methods comprise administering to a subject (inneed thereof) any one (or more) of the compounds, or compositionscomprising the compounds, described herein, wherein ion movement(particularly chloride ion conductance or current) by CaCC is inhibited.A subject includes a human and non-human animal. Non-human animals thatmay be treated include mammals, for example, non-human primates (e.g.,monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice,gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig,miniature pig), equine, canine, feline, bovine, and other domestic,farm, and zoo animals. In certain embodiments, methods for treatingsecretory diarrheas are provided herein, comprising administering to asubject any one (or more) aminothiophene or aminothiazole compoundsdescribed herein (or compositions comprising the any one or morecompounds) in combination with an agent that inhibits CFTR. Exemplarycompounds that may be used to inhibit CFTR include, but are not limited,to thiazolidinone compounds and hydrazide compounds (see, e.g.,thiazolidinone compounds (e.g.,3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTRinh-172)) and hydrazide compounds (see, e.g.,U.S. Pat. No. 7,235,573; U.S. Patent Application Publication No.2005-0239740; Muanprasat et al., J. Gen. Physiol. 124:125-37 (2004); Maet al., J. Clin. Invest. 110:1651-58 (2002)).

The aminothiophene compounds having the structure of formula I andsubstructures of formula Ia-Ii and aminothiazole compounds having thestructure of formula II and substructures of formula IIa-IIh, asdescribed herein, may be formulated in a pharmaceutical composition foruse in treatment, which includes preventive treatment, of a disease ordisorder manifested by increased intestinal fluid secretion, such assecretory diarrhea or that is associated with excess (i.e., abnormallyincreased) mucus secretion (i.e., abnormally increased mucus secretionis a condition associated with or is a sequelae of the disease ordisorder). A pharmaceutical composition may be a sterile aqueous ornon-aqueous solution, suspension or emulsion, which additionallycomprises a physiologically acceptable excipient (also called apharmaceutically acceptable or suitable excipient or carrier) (i.e., anon-toxic material that does not interfere with the activity of theactive ingredient). Such compositions may be in the form of a solid,liquid, or gas (aerosol). Alternatively, compositions described hereinmay be formulated as a lyophilizate, or compounds may be encapsulatedwithin liposomes using technology known in the art. Pharmaceuticalcompositions may also contain other components, which may bebiologically active or inactive. Such components include, but are notlimited to, buffers (e.g., neutral buffered saline or phosphate bufferedsaline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans),mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, chelating agents such as EDTA or glutathione, stabilizers,dyes, flavoring agents, and suspending agents and/or preservatives.

Any suitable excipient or carrier known to those of ordinary skill inthe art for use in pharmaceutical compositions may be employed in thecompositions described herein. Excipients for therapeutic use are wellknown, and are described, for example, in Remington: The Science andPractice of Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa.(2005)). In general, the type of excipient is selected based on the modeof administration. Pharmaceutical compositions may be formulated for anyappropriate manner of administration, including, for example, topical,oral, nasal, intrathecal, rectal, vaginal, intraocular, subconjunctival,sublingual or parenteral administration, including subcutaneous,intravenous, intramuscular, intrasternal, intracavernous, intrameatal orintraurethral injection or infusion. For parenteral administration, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above excipients or a solidexcipient or carrier, such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, kaolin, glycerin, starchdextrins, sodium alginate, carboxymethylcellulose, ethyl cellulose,glucose, sucrose and/or magnesium carbonate, may be employed.

A pharmaceutical composition (e.g., for oral administration or deliveryby injection) may be in the form of a liquid. A liquid pharmaceuticalcomposition may include, for example, one or more of the following: asterile diluent such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils that may serve as the solvent or suspending medium, polyethyleneglycols, glycerin, propylene glycol or other solvents; antibacterialagents; antioxidants; chelating agents; buffers and agents for theadjustment of tonicity such as sodium chloride or dextrose. A parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. The use of physiological saline ispreferred, and an injectable pharmaceutical composition is preferablysterile.

A composition comprising an aminothiophene compound having the structureof formula I and substructures of formula IA, Ia-Ii and aminothiazolecompound having the structure of formula II and substructures of formulaIIa-IIh may be formulated for sustained or slow release. Suchcompositions may generally be prepared using well known technology andadministered by, for example, oral, rectal or subcutaneous implantation,or by implantation at the desired target site. Sustained-releaseformulations may contain an agent dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.Excipients for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. The amount of activecompound contained within a sustained release formulation depends uponthe site of implantation, the rate and expected duration of release, andthe nature of the condition to be treated or prevented.

The dose of the composition for treating a disease or disorderassociated with aberrant CaCC function, including but not limited tointestinal fluid secretion, secretory diarrhea, such as a toxin-induceddiarrhea, or secretory diarrhea associated with or a sequelae of anenteropathogenic infection, Traveler's diarrhea, ulcerative colitis,irritable bowel syndrome (IBS), AIDS, chemotherapy and diseases orconditions described herein associated with overproduction or excesssecretion of mucus (e.g., asthma, chronic obstructive pulmonarydisorder, bronchiectasis) may be determined according to parametersunderstood by a person skilled in the medical art. Accordingly, theappropriate dose may depend upon the subject's condition, that is, stageof the disease, general health status, as well as age, gender, andweight, and other factors considered by a person skilled in the medicalart.

Pharmaceutical compositions may be administered in a manner appropriateto the disease or disorder (or condition) to be treated as determined bypersons skilled in the medical arts. An appropriate dose and a suitableduration and frequency of administration will be determined by suchfactors as the condition of the patient, the type and severity of thepatient's disease, the particular form of the active ingredient, and themethod of administration. In general, an appropriate dose (or effectivedose) and treatment regimen provides the composition(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit (e.g., animproved clinical outcome, such as more frequent complete or partialremissions, or longer disease-free and/or overall survival, improvedquality of life, or a lessening of symptom frequency and/or severity).

Consistent with the understanding in the medical arts, treatment ortreating refers to the medical management of a disease or disorder.Clinical assessment of the level of dehydration and/or electrolyteimbalance may be performed to determine the level of effectiveness of acompound and whether dose or other administration parameters (such asfrequency of administration or route of administration) should beadjusted. In addition, particularly with respect to chronic diseasessuch as cystic fibrosis, asthma, COPD, and bronchiectasis, clinicalassessment of improvement may be determined by lessening of symptomfrequency and/or severity. For example, clinical assessment may includedetermining the number of hospitalizations, particularly resulting fromexacerbations related to bacterial infections. Clinical evaluationspatients with such chronic diseases also includes quality of lifeassessment. Therefore, a patient or subject, particularly a patient thathas a chronic disease such as cystic fibrosis, COPD, or bronchiectasis,for example, may be treated by administering to the subject thecompounds and compositions described herein, and as such the treatmenthas a therapeutic or prophylactic benefit.

Optimal doses may generally be determined using experimental modelsand/or clinical trials. The optimal dose may depend upon the body mass,weight, or blood volume of the subject. In general, the amount of asmall molecule compound as described herein, that is present in a dose,ranges from about 0.01 μg to about 1000 μg per kg weight of the host.The use of the minimum dose that is sufficient to provide effectivetherapy is usually preferred. Subjects may generally be monitored fortherapeutic effectiveness using assays suitable for the condition beingtreated or prevented, which assays will be familiar to those havingordinary skill in the art and are described herein.

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures. The following examples are providedmerely as illustrative of various embodiments and shall not be construedto limit the invention in any way.

EXAMPLES Example 1 Cell-Based Assay to Identify Calcium-ActivatedChloride Channel Inhibitors

HT-29 cells (ATCC HTB-38) were obtained from the American Type CultureCollection (Manassas, Va.) and grown in DMEM supplemented with 10% fetalbovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.T84 cells were maintained in DMEM/F12 (1:1) medium containing 10% FBS,100 units/ml penicillin and 100 μg/ml streptomycin. Fisher rat thyroid(FRT) expressing human CFTR and YFP-H148Q/I152L were generated asdescribed (Ma et al., J. Clin. Invest. 110:1651-8 (2002)), and grown inF-12 Modified Coon's medium supplemented with 10% FBS, 2 mM glutamine,100 units/ml penicillin, and 100 μg/ml streptomycin. All cells weregrown at 37° C. in 5% CO₂/95% air.

HT-29 cells were infected with a lentivirus encoding YFP-H148Q/I152Laccording to molecular biology methods routinely practiced by personsskilled in the art. Prior to infection, HT-29 cells were cultured in90-mm diameter plates until ˜80% confluent in McCoy's 5a mediumsupplemented with 1.5 mM L-glutamine, 10% FBS, and 2.2 g/L sodiumbicarbonate. The cells were washed three times with PBS, and then 1 mlof high-titer lentiviral supernatant was added to each well in thepresence of 8 μg/ml polybrene. The cells were incubated at 37° C. in 5%CO₂/95% air for 6 hours, and medium was then replaced with the regularDMEM growth medium described above. YFP expression was detected 48 hafter infection. As depicted in FIG. 1B, HT-29 cells stably expressingYFP-H148Q/I152L were brightly fluorescent, with nearly all cells havingfluorescence. This particular YFP indicator (YFP-H148Q/I152L) used forscreening was 50% quenched by ˜3 mM iodide.

For experiments with thapsigargin or calcimycin, cells in 96-well platesat 100% confluence were washed 3 times with PBS, incubated with testcompounds at 32.5 μM for 5 min in PBS in a final volume of 60 μl/well,and then incubated with 2 μM thapsigargin for 7 min or 10 μM calcimycinfor 3 min before assay of iodide influx.

Several CaCC agonists were assayed to establish the cellular model forCaCC inhibitor screening. Histamine (100 μM), calcimycin (10 μM), ATP(100 μM), carbachol (100 μM) and forskolin (10 μM) were testedindividually (FIG. 2A) and in combinations (FIG. 2B). The CFTR inhibitorCFTR_(inh)-172 (20 μM) was also tested. Of the agonists testedindividually, carbachol and ATP produced the strongest responses asindicated by the slopes of the fluorescence decrease followingextracellular iodide addition. In combination, carbachol and ATPproduced the greatest response observed for the combinations. Increasediodide influx was not observed following forskolin addition, nor didCFTR_(inh)-172 inhibit iodide influx in response to calcium agonists,indicating that the cells used in the assay expressed little or nofunctional CFTR. A combination of carbachol and ATP was selected forcompound screening.

Iodide influx measurements were done to establish agonist concentrationsand addition times. Concentration dependence studies for carbachol (FIG.2C) and ATP indicated maximal responses at 100 μM. FIG. 2D shows reducediodide influx as a function of time between addition of 100 μMcarbachol/ATP and extracellular iodide, which is a consequence of thetransient elevation in cytoplasmic calcium produced by these agonists.The greatest iodide influx was observed when agonists were added at ˜10s prior to iodide; however, the influx was not much greater than whenagonists were added at the time of iodide addition. Therefore, forhigh-throughput screening, in part, to simplify assay conditions,agonists (carbachol and ATP, each 100 μM) were added together withiodide.

Compounds for primary screening were purchased from ChemDiv (San Diego,Calif.). The compound collection contained 50,000 diverse, drug-likecompounds with >90% of compounds in the molecular size range 200-450daltons. Compounds for the secondary screening were purchased fromChemDiv and Asinex. Compounds were prepared in 96-well plates(Corning-Costar) as 10 mM solutions in dimethylsulfoxide (DMSO). For theprimary screen compounds were tested in groups of 4 compounds per well.The compounds were screened at a concentration 32.5 μM. All chemicalswere purchased from SIGMA-ALDRICH (St. Louis, Mo.).

For high-throughput screening, iodide influx was measured in theYFP-expressing HT-29 cells in a 96-well format using an automatedworkstation capable of assaying more than 10,000 compounds overnight.Screening was performed using a customized screening system (BECKMANCOULTER, Inc., Indianapolis, Ind.) consisting of the SAGIAN Core systemintegrated with SAMI software, and equipped with an ORCA arm for labwaretransport, a 96-channel head BIOMEK FX, CO₂ incubator, plate washer, barcode reader, delidding station, and two FLUOstar OPTIMA fluorescenceplate readers (BMG Labtechnologies, Durham, N.C.), each equipped withsyringe pumps and custom excitation/emission filters (500/544 nm;CHROMA, Brattleboro, Vt.).

HT-29 cells expressing YFP-H148Q/I152L were plated in 96-well plates at70% confluence. Plates were incubated overnight at 37° C., 5% CO₂ andthen the growth medium was replaced with fresh growth medium. Cells wereincubated further until 95% confluence (12-18 h), washed 3 times withPBS, and incubated with the test compounds at 32.5 μM finalconcentration for 5 min in PBS, in a final volume of 60 μl/well. YFPfluorescence was measured 1 s before and for 30 s after addition of aPBS-iodide solution (PBS that has 100 mM chloride replaced by iodide)containing carbachol and ATP (100 each). Each 96-well plate contained‘positive’ controls (DMSO vehicle without agonists or test compounds)and ‘negative’ controls (DMSO vehicle with agonists but without testcompounds). In some experiments solutions included (individually or incombination): carbachol, ATP, or histamine (at 50, 100, 150 or 200 μM),calcimycin (10 μM), thapsigargin (2 μM) and CFTR_(inh)-172 (20 μM).

Iodide influx (d[I⁻]/dt at t=0) was computed from fluorescence timecourse data by single exponential regression, as described (Ma et al.,J. Clin. Invest. 110:1651-8 (2002)). Percentage inhibition was computedas: percentageinhibition=100×(Slope_(negative control)−Slope_(test compound))/(Slope_(negative control)−Slope_(positive control)).

FIG. 3A (left) shows representative YFP fluorescence kinetics measuredin single wells of 96-well plates. Each curve consisted of recording ofbaseline fluorescence for 1 second, followed by 30 seconds of continuousrecording of fluorescence after rapid addition of a solution containingiodide and the CaCC agonist combination (carbachol and ATP, each 100μM). Following a small solution addition artifact, there was littledecrease in fluorescence in the absence of activators, compared to arobust reduction in fluorescence with agonists. FIG. 3A (right) shows afrequency histogram of iodide influx rates, d[f]/dt, in individual wellsfor positive (no agonists) or negative (with agonists) controls. Thecomputed Z′-factor for the assay was very good, 0.74, indicatingadequacy of a single compound screening to identify putative CaCCinhibitors.

CaCC inhibitors were identified based on inhibition of ATP/carbacholstimulated iodide-influx. Screening yielded six classes of putative CaCCinhibitors, two of which, 3-acyl-2-aminothiophenes and5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cellsin response to multiple calcium-elevating agonists. FIG. 3B (left) showsexamples of data from single wells for compounds with ‘strong’, ‘weak’and no inhibition activity. FIG. 3B (right) summarizes percentageinhibition data as a frequency histogram. Of 50,000 small moleculesscreened (in groups of four), 300 compound groups had CaCC inhibitoryactivity as defined by a 70% cutoff (vertical dashed line). Retesting ofthese 300 compound groups indicated a false-positive rate of ˜40%.

Further analysis was performed on fifteen-compound groups that producedgreatest inhibition when retested at 10 and 30 μM concentrations. Thecompound responsible for activity in each group was determined bytesting individual compounds in each group. In each case, a singleactive compound was identified in the groups of four. FIG. 3C showsstructures of the most active of six classes of putative CaCC inhibitors(classes A-F) identified by single compound testing; in some cases,particularly for classes A and B, similar structures were seen severaltimes. Percentage inhibition of these compounds at 30 μM was in therange 60 to >90%. Based on multiple criteria, including potency, watersolubility, drug-like properties, identification of active analogs,chemical stability and CaCC targeting, compounds of classes A and B wereanalyzed further. Class A and B compounds were resynthesized, analyzedfor mechanism-of-action, and assessed for ‘druggability’ by determiningstructure-activity relationships.

Example 2 Synthesis of Candidate CaCC Inhibitor Compounds

This example describes synthesis of exemplary class A and B compounds tohigh purity and verified their structures and chemical stability inaqueous solutions.

CaCC_(inh)-A01:

The synthesis of CaCC_(inh)-A01 was accomplished in three steps (FIG.4A), involving Knovenagle condensation of methyl cyanoacetate witht-butylcyclohexanone followed by cyclization on elemental sulfur.Purification by flash chromatography afforded the 2-aminothiopheneGewald product. Acyclation of the aminothiophene gave the N-acyl methylester [2] in good yield. Ester [2] was hydrolyzed with NaOH to giveCaCC_(inh)-A01, which was purified by chromatography andrecrystallization.

In greater detail, the synthesis of6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid [1] began with a mixture of 4-(t-butyl) cyclohexanone (0.154 g,1.00 mmol), methyl-2-cyanoacetate (0.109 g, 1.1 mmol), morphiline (0.104g, 1.2 mmol) and elemental sulfur (0.048 g, 1.5 mmol) in methanol (5mL), which was microwaved for 10 min at 120° C. using a Biotagemicrowave reactor. Purification by flash chromatography afforded [1](0.242 g, 0.91 mmol) in 91% yield (Gewald et al., J Prakt. Chem.311:402-407 29 (1969); Sridhar et al., Tetrahedron Lett. 48:3171-72(2007)). Acylation of the amine with furfuryl chloride (0.186 mg, 0.70mmol) gave compound [2] in 87% yield. Ester hydrolysis of [2] (0.179 g,0.500 mmol) was accomplished with NaOH in methanol affordingt-butylbenzothiophene (CaCC_(inh)-A01). CaCC_(inh)-A01 was purified bycolumn chromatography (160 mg, 93% yield). ¹H NMR (400 MHz, CDCl₃):δ12.88 (s, 1H), 7.59 (s, 1H), 7.30 (d, J=3.2 Hz, 1H), 6.59 (dd, J=7.6 Hzand 2.0 Hz, 1H), 3.18 (d, 2H), 2.72 (m, 2H), 2.43 (t, 1H), 2.05 (m, 1H),1.51 (m, 1H), 1.35 (m, 1H), 0.95 (s, 9H); ¹³C NMR (CDCl₃): δ 179.2,170.6, 154.8, 149.1, 146.8, 145.4, 131.8, 128.3, 116.6, 112.9, 94.5,45.1, 32.6, 27.4, 26.0, 24.5; LC-MS: m/z 346.16 [M+H]⁺ (Nova-Pak C₁₈column, 99%, 200-400 nm).

CaCC_(inh)-B01:

The synthesis of CaCC_(inh)-B01 also involved three steps (FIG. 4B).Reaction of 4-amino-2-hydroxybenzoic acid with thiophosgene in HCl gaveisothiocyanate in good yield. Treatment of isothiocyanate with ammoniumhydroxide gave thiourea, which was then reacted with2-bromo-1-p-tolylethanone to give CaCC_(inh)-B01.

In greater detail, the synthesis of2-hydroxy-4-(4-p-tolylthiazol-2-ylaminobenzoic acid [4] began with thesynthesis of thiourea [5]. Reaction of thiophosgene (2.7 g, 23.890 mmol)with 4-amino-2-hydroxybenzoic acid (3.06 g, 20 mol) in aqueous HCl (41mL) afforded thioisocyanate by crystallization in 83% yield (Seligman etal., J. Am. Chem. Soc. 75:6334-35 (1953)). Treatment of thioisocyanatewith ammonium hydroxide gave thiourea in 84% yield. Thiazole cyclizationof thiourea [5] (0.300 g, 1.415 mmol) with 2-bromo-1-p-tolylethanone(0.298 g, 1.415 mmol) in EtOH (15 mL) gave aminothiazole. Theaminothiazole was purified by column chromatography to affordCaCC_(inh)-B01 (406 mg, 88% yield). ¹H NMR (400 MHz, CDCl₃): δ10.67 (s,1H), 7.80 (d, J=8.0 Hz, 2H), 7.71 (d, J=8.1 Hz, 1H), 7.58 (d, J=2.0 Hz,1H), 7.36 (s, 1H), 7.25 (d, J=8.0, 2H), 7.04 (dd, J=8.1 Hz and 2.0 Hz,1H), 2.49 (s, 1H), 2.31 (s, 3H); ¹³C NMR (CDCl₃): δ 171.7, 162.7, 162.0,150.3, 147.2, 137.2, 131.7, 131.3, 129.3, 125.6, 108.5, 105.4, 103.6,103.1, 20.8; LC-MS: m/z 327.11 [M+H]⁺ (Nova-Pak C₁₈ column, 97%, 200-400nm).

CaCC_(inh)-A01 and CaCC_(inh)-B01 were confirmed by ¹H-NMR, ¹³C-NMR, andmass spectrometry. The aqueous solubility of CaCC_(inh)-A01 andCaCC_(inh)-B01 in PBS was >500 μM, as measured by optical absorbance ofa saturated solution after appropriate dilution. The high aqueoussolubility is a consequence of their polarity and negative charge atphysiological pH. By liquid chromatography compound purity was >97% and99% for CaCC_(inh)-A01 and CaCC_(inh)-B01, respectively.

Testing of the purified compounds using the fluorescence plate readerassay verified their activities, with IC50 values ˜10 μM (FIG. 4C).These apparent IC50 values are only semi-quantitative because of themulti-step nature of the screening assay and because of the ˜3-foldcompound dilution at the time of iodide addition during the assay.Accurate IC50s were determined by electrophysiological assays asdescribed herein.

Example 3 Confirmation of CaCC as the Target of Class A and B Compounds

This Example describes analysis of Class A and B compounds to confirmtheir activity was specific for CaCCs. As shown in the schematic of thescreening strategy illustrated in FIG. 1A, the screening assay couldidentify other target interactions such as ligand-receptor binding,calcium elevation, and calcium-calmodulin CaMKII) signaling. Todistinguish between pre- and post-calcium signaling targets, compoundsof each class were tested following stimulation of HT-29 cells bythapsigargin, which produces calcium elevation without ligand-receptorbinding or phosphoinositide signaling. FIG. 5A shows that each of thecompounds inhibited iodide influx following thapsigargin, except for theclass E compounds, whose target is thus likely upstream from calciumsignaling.

For experiments with thapsigargin or calcimycin, cells in 96-well platesat 100% confluence were washed 3 times with PBS, incubated with testcompounds at 32.5 μM for 5 min in PBS in a final volume of 60 μl/well,and then incubated with 2 μM thapsigargin for 7 min or 10 μM calcimycinfor 3 min before assay of iodide influx.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Experiments were performed to determine whether the compounds affectedCFTR because CFTR is a major intestinal chloride channel and evidenceexists for cross-talk between cAMP and calcium signaling in intestinalcell chloride secretion (Schultheiss et al., Eur. J. Pharmacol.546:161-70 (2006); Takahashi et al., J. Med. Microbiol. 49:801-10(2000); Chao et al., EMBO J. 13: 1065-72 (1994)). As described (Ma etal., J. Clin. Invest. 110:1651-8 (2002)), FRT cells expressing humanwildtype CFTR and YFP-H148Q/I152L, at 100% confluence, were washed 3times with PBS, leaving 60 μL. CFTR was activated by a cocktailcontaining forskolin (20 μM), genistein (50 μM), andisobutylmethylxanthine (100 μM). Iodide influx as described in Example 1was measured at 10 min after addition of test compounds by determiningYFP fluorescence for 2 s before and 20 s after addition of 165 μl ofPBS-iodide. FIG. 5B shows no CFTR was not inhibited by any of thecompounds at 30 μM; CFTR_(inh)-172 inhibition shown is the positivecontrol.

Example 3 Confirmation of CaCC as the Target of Class A and B Compounds

This Example describes analysis of Class A and B compounds to confirmthat their activity was specific for CaCCs. As shown in the schematic ofthe screening strategy illustrated in FIG. 1A, the screening assay couldidentify other target interactions such as ligand-receptor binding,calcium elevation, and calcium-calmodulin CaMKII) signaling. Todistinguish between pre- and post-calcium signaling targets, compoundsof each class were tested following stimulation of HT-29 cells bythapsigargin, which produces calcium elevation without ligand-receptorbinding or phosphoinositide signaling. FIG. 5A shows that each of thecompounds inhibited iodide influx following thapsigargin, except for theclass E compounds, whose target is thus likely upstream from calciumsignaling.

For experiments with thapsigargin or calcimycin, cells in 96-well platesat 100% confluence were washed 3 times with PBS, incubated with testcompounds at 32.5 μM for 5 min in PBS in a final volume of 60 μl/well,and then incubated with 2 μM thapsigargin for 7 min or 10 μM calcimycinfor 3 min before assay of iodide influx.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Experiments were performed to determine whether the compounds affectedCFTR because CFTR is a major intestinal chloride channel and evidenceexists for cross-talk between cAMP and calcium signaling in intestinalcell chloride secretion (Schultheiss, et al., Eur. J. Pharmacol.546:161-70 (2006); Takahashi, et al., J. Med. Microbiol. 49:801-10(2000); Chao, et al., EMBO J. 13:1065-72 (1994)). As described (Ma etal., J. Clin. Invest. 110:1651-8 (2002)), FRT cells expressing humanwildtype CFTR and YFP-H148Q/I152L, at 100% confluence, were washed 3times with PBS, leaving 60 μL. CFTR was activated by a cocktailcontaining forskolin (20 μM), genistein (50 μM), andisobutylmethylxanthine (100 μM). Iodide influx as described in Example 1was measured at 10 min after addition of test compounds by determiningYFP fluorescence for 2 s before and 20 s after addition of 165 μl ofPBS-iodide. FIG. 5B shows no CFTR was not inhibited by any of thecompounds at 30 μM; CFTR_(inh)-172 inhibition shown is the positivecontrol.

[Ca²⁺]_(i) Measurements

Cytoplasmic calcium was measured to determine whether CaCC_(inh)-A01 andCaCC_(inh)-B01 interfered with agonist-induced calcium signal in HT-29cells. [Ca²]_(i) was measured in confluent monolayers of HT-29 cellsafter loading with Fura-2 (2 μM Fura-2-AM, 30 min, 37° C.). Labeledcells were mounted in a perfusion chamber on the stage of an invertedepifluorescence microscope. Cells were superfused with (in mM): 140NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 D-glucose, and 10 HEPES (pH 7.4),initially without and then with ATP/carbachol. Test compounds werepresent in some experiments for 10 min prior to agonist addition. Fura-2fluorescence was recorded at excitation wavelengths of 340 nm and 380 nmusing standard procedures

FIG. 5C shows [Ca²⁺]_(i) elevations in response to 1 μM ATP, 100 μM ATP,and 100 μM carbachol (left), or 1 μM ionomycin (middle). Pretreatmentwith CaCC_(inh)-A01 or CaCC_(inh)-B01 at high concentration prior toagonist addition did not significantly reduce the ATP orcarbachol-induced [Ca²⁺]_(i) elevations (right).

CaMKII

Regulation of CaCC activation by CaMKII occurs in a cell type-dependentmanner (Hartzell et al., Annu. Rev. Physiol. 67:719-58 (2005)). CaMKIIhas been reported to regulate calcium-activated chloride current inHT-29 and T84 cells (Worrell et al., Am. J. Physiol. 260:C877-82 (1991);Morris et al., Am. J. Physiol. 264:C977-85 (1993); Chan et al., J. Biol.Chem. 269:32464-8 (1994); Braun et al., J. Physiol. 488:37-55 (1995)).To determine whether the CaCC inhibitors interfered withATP/carbachol-induced CaMKII activation, CaMKII phosphorylation in HT-29cells was measured by immunoblot analysis. Calcium/calmodulin-dependentprotein kinase II (CaMKII) was activated by 2 min treatment of HT-29cells with ATP/carbachol (each 100 μM). Cells were then lysed with celllysis buffer (20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 2mM EDTA, 50 mM α-glycerolphosphate, 1 mM Na₃VO₄, 1 mM DTT, and completeprotease inhibitor mixture (Roche Applied Science)). Cell debris wasremoved by centrifugation, and proteins in the supernatant were resolvedby SDS-PAGE and immunoblotted using standard procedures (transfer toPDVF membrane, 1 h blocking in 5% nonfat dry milk, primary/secondaryantibody incubations, enhanced chemiluminescence detection). Rabbitpolyclonal antibodies for anti-phospho-CaMKII (Thr286) and β-actin werepurchased from Cell Signaling Technology (Danvers, Mass.).

FIG. 5D present an immunoblot demonstrating the presence of pCaMKIIimmunoreactivity following agonist treatment. Pretreatment with 30 μMCaCC_(inh)-A01 or CaCC_(inh)-B01 did not significantly affectagonist-induced CaMKII phosphorylation. β-actin is shown as loadingcontrol.

Example 4 Effect of Class A and B Compounds on Calcium-DependentChloride Current

Patch Clamp—Calcium Dependent Chloride Current

Whole-cell patch-clamp was performed to investigate inhibition ofcalcium-dependent chloride current in HT-29 cells by CaCC_(inh)-A01 andCaCC_(inh)-B01. Whole cell recordings were accomplished with HT-29 cellsat room temperature. The pipette solution contained (in mM): 30 CsCl,100 Cs-aspartate, 1 MgCl₂, 0.5 EGTA, 2 Tris-ATP, and 10 HEPES (pH 7.2with CsOH), and the bath solution contained (in mM) 140N-methyl-D-glucamine chloride (NMDG-Cl), 1 CaCl₂, 1 MgCl₂, 10 glucose,and 10 HEPES (pH 7.2). In one set of studies, symmetric NMDG-Clsolutions contained 140 mM NMDG-Cl, 1 mM MgCl₂, 0.5 mM EGTA, 2 mMTris-ATP, and 10 mM HEPES, pH 7.2. Pipettes were pulled fromborosilicate glass and had resistances of 3-5 MS2 after fire polishing.Seal resistances were typically between 3-10 GΩ. After establishing thewhole cell configuration, CaCCs were activated by 1 μM ionomycin. Wholecell currents were elicited by applying hyperpolarizing and depolarizingvoltage pulses from a holding potential of 0 mV to potentials between−120 mV and +120 mV in steps of 20 mV. Currents were filtered at 5 kHz,and digitized and analyzed using an AxoScope 10.0 system and a Digidata1440A AC/DC converter (Molecular Devices, Sunnyvale, Calif.). Meancurrents were expressed as current densities (picoampere per picofarad(pA/pF)).

Treatment with 1 μM ionomycin produced large currents of 28±3 pA/pF (Vm+100 mV) in NMDG-Cl solutions, with outwardly rectifying I-Vrelationship as shown in FIGS. 6A and B). Ionomycin-stimulated currentswere measured when the current was maximally activated at a Vm of −40mV. As summarized in FIG. 6C, calcium-dependent chloride current wasreduced by 38±14%, 66±10%, and 91±1% by 0.1, 1 and 10 μM CaCC_(inh)-A01,respectively, and by 11±7%, 34±12%, and 77±4% by 0.1, 1 and 10 μMCaCC_(inh)-B01. As a control to exclude interference by transientreceptor potential and nonselective cation channels, ionomycin-inducedchloride currents were also recorded in the whole-cell configurationusing symmetric NMDG-Cl solutions. The results are presented in FIG. 6D.As predicted, pretreatment with CaCC_(inh)-A01 and CaCC_(inh)-B01reduced ionomycin-induced currents.

Short Circuit Current Measurements (Chloride Secretion)

Chloride secretion was measured in T84 cells to investigate whether theCaCC inhibitors identified from screening of HT-29 cells also inhibitedthe CaCC in a different human intestinal cell line. ATP/carbachol wereused to induce calcium-dependent chloride secretion inwell-differentiated T84 cell monolayers. T84 cells were seeded at adensity of 10⁵ cm⁻² on permeable supports (Snapwell, 1.12 cm² surfacearea) and grown until confluent. Supports containing confluent cellmonolayers were mounted in Snapwell diffusion chambers. Cells werebathed for a 30 min stabilization period in HCO₃ ⁻-buffered solutioncontaining (mmol/L): 120 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 D-glucose, 5HEPES, and 25 NaHCO₃ (pH 7.4), aerated with 95% O₂/5% CO₂ at 37° C.Monolayers were voltage-clamped at 0 mV (EVC4000 Multi-Channel V/IClamp, World Precision Instruments), and short-circuit current (Ise) wasrecorded continuously with agonists/inhibitors added at specified times.

Application of carbachol followed ATP after a recovery period producedlarge short-circuit current as shown in FIG. 7A. Carbachol andATP-induced short-circuit currents are summarized in FIG. 7B. Initialcarbachol-induced currents were similar, as expected, indicated similarchloride secretory activities of the cultures. ATP-induced shortcircuit-currents were reduced by 38±7% and 78±3% at 10 and 30 μMCaCC_(inh)-A01, respectively, and by 29±11% and 64±3% by 10 and 30 μMCaCC_(inh)-B01.

The aminothiophenes and aminothiazoles identified in the screen andanalyzed in the experiments described above did not interfere withagonist-induced cytoplasmic calcium elevation or calmodulin (CaMKII)phosphorylation. As indicated by patch-clamp analysis, the compoundsthat were tested inhibited CaCC gating. These results confirm that thephenotype-based small molecule screen identified novel chemical classesof inhibitors of CaCC chloride conductance that target the CaCC itselfrather than upstream signaling mechanisms and/or any of the othermechanisms tested.

Example 4 Effect of Class A and B Compounds on Calcium-DependentChloride Current

This Example describes the structure activity relationship of theaminothiophene compounds (i.e., having a structure of formula I orsubstructures thereof) and aminothiazole compounds (i.e., having astructure of formula II or substructures thereof). SAR analysis wasperformed on 936 commercially available aminothiophenes (class Aanalogs) and 944 aminothiazoles (class B analogs). Tables 3 and 4provide semi-quantitative CaCC inhibition data (from plate reader assay,see Example 1) for 18 active aminothiophenes (class A) and 14 activeaminothiazoles (class B).

TABLE 3 Structure-Activity of Aminothiophene (Class A) CaCC Inhibitors %inhibi- tion at Compound R¹ R² R³ n 25 μM CaCC_(inh)-A01 t-butyl —OH

1 100  CaCC_(inh)-A02 t-butyl —OH

1 95 CaCC_(inh)-A03 t-butyl —OH

1 50 CaCC_(inh)-A04 t-butyl —OH

1 85 CaCC_(inh)-A05 t-butyl —OH

1 75 CaCC_(inh)-A06 t-butyl —OCH₂CH₃

1 75 CaCC_(inh)-A07 t-butyl —OCH₂CH₃

1 98 CaCC_(inh)-A08 t-butyl —OCH₂CH₃

1 80 CaCC_(inh)-A09 t-butyl —OCH₃

1 45 CaCC_(inh)-A10 t-pentyl —OCH₂CH₃

1 75 CaCC_(inh)-A11 t-butyl —OCH₃

1 81 CaCC_(inh)-A12 H —OH

2 15 CaCC_(inh)-A13 H —OH

2 27 CaCC_(inh)-A14 H —OH

2 18 CaCC_(inh)-A15 H —OH

1 20 CaCC_(inh)-A16 t-butyl

1 42 CaCC_(inh)-A17 H —OH

1 07 CaCC_(inh)-A18 t-butyl

1 35

A common moiety in compounds that exhibited the highest inhibitoryactivity in each compound class was the presence of a carboxylic acidfunctional group, which is also present in certain other known chloridechannel inhibitors.

Class A compounds (aminothiophene) that had t-butyl at position R¹conferred the greatest inhibition; inhibition was reduced when R¹ was H(e.g., CaCC_(inh)-A12-A15) and reduced when R¹ was methyl. Whilecompounds having a cyclohexyl ring system had greater activity thancompounds with cyclopentyl and cycloheptyl (varying n) rings (i.e., thering to which R¹ is attached), several cycloheptylthiophenes wereactive. At R², hydroxy, methoxy, ethoxy and substituted phenyl amideswere identified. Compounds with a methyl ester or an ethyl ester at R²were active, and certain of these compounds identified also had an alkylcarboxylic acid present at R³ (see, e.g., CaCC_(inh)-A06-A11). Withrespect to the moiety comprising R³ (—NHC(═O)R³), activity was observedfor compounds comprising substituted phenyl, substituted heterocycles,and substituted alkyl. The most active compound that had substitutedalkyl at R³ was the analog comprising trans-3-carboxyacrylamido(CaCC_(inh)-A07). The presence of substituted phenyl at R³ in compoundswas observed. A variety of compounds with heterocycles at R³ werescreened; active compounds comprising a furyl substituent wereidentified, and the most potent compound identified was(CaCC_(inh)-A01).

TABLE 4 Structure-Activity of Aminothiazole (Class B) CaCC inhibitors %inhibition at Compound R⁷ R⁸ R⁹ R¹⁰ 25 μM CaCC_(inh)-B01

H H 4-methyl 100  CaCC_(inh)-B02

—CH₂CO₂H H 4-methyl 40 CaCC_(inh)-B03

—CH₂CO₂H H 4-methyl 60 CaCC_(inh)-B04

—CH₂CO₂H H 4-methyl 65 CaCC_(inh)-B05

—CH₂CO₂H H 4-methyl 60 CaCC_(inh)-B06

n-propyl 2-methyl 4-methyl 100  CaCC_(inh)-B07

—CH₂CO₂H 2-methyl 4-methyl 70 CaCC_(inh)-B08

—CH₂CO₂H 2-methyl 4-methyl 75 CaCC_(inh)-B09

H H 4-phenoxy 65 CaCC_(inh)-B10

H H 3-CF₃ 55 CaCC_(inh)-B11

—CH₂CO₂H H 4-methyl 65 CaCC_(inh)-B12

phenyl H 4-methyl 03 CaCC_(inh)-B13 —CH₂CH₃ H H 4-methyl 20CaCC_(inh)-B14

—(CH₂)₂CO₂H H 4-methoxy 18

With respect to aminothiazole compounds, the substituent at R¹⁰ that wasobserved in a majority of active compounds was a methyl substitution atthe 4-position of the aromatic ring (see, e.g., CaCC_(inh)-B01-B06).Certain active analogs of such compounds had an additional methylsubstitution at the 2-position (see, e.g., CaCC_(inh)-B07-B09). Asobserved with aminothiophene compounds, aminothiazole compounds that hada carboxylic acid functional group were most active. Examples of suchaminothiazole compounds had carboxy-substituted alkyl at R⁸ or a carboxysubstituted phenyl at R⁷. At position R⁸, active compounds containedhydrogen, propyl, or an acetic acid group (—(CH₂)_(n)C(═O)OH). At theposition R⁷, a variety of substituted phenyl groups were tolerated.Electron donating substituents produced inactive compounds.

All the above U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications, andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety.

From the foregoing, although specific embodiments of the invention havebeen described herein for purposes of illustration, variousmodifications may be made without deviating from the spirit and scope ofthe invention. Those skilled in the art will recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A composition comprising a physiologically acceptable excipient and acompound having the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein R¹ is hydrogen or optionally substituted alkyl; R² is hydroxy,optionally substituted alkoxy, or optionally substituted phenylamino; R³is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted cycloalkyl, optionally substituted phenyl, oroptionally substituted heterocyclyl; and n is 0, 1, or 2, and whereinthe compound of structure I comprises at least one —COON.
 2. Thecomposition of claim 1 wherein the compound has the following structureI(A):

wherein n is 1 or
 2. 3. The composition of claim 1 wherein n is 1 and R¹is hydrogen, tert-butyl, or tert-pentyl, and the compound has thefollowing structure (Ia), (Ib), or (Ic):

4-25. (canceled)
 26. A composition comprising a physiologicallyacceptable excipient and a compound having the following structure (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein R⁷ is optionally substituted C₁₋₆ alkyl, optionally substitutedphenyl, or optionally substituted phenylacyl; R⁸ is hydrogen, optionallysubstituted C₁₋₆ alkyl, or optionally substituted phenyl; R⁹ and R¹⁰ arethe same or different and independently hydrogen, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substituted phenoxy.27. The composition of claim 26 wherein at least one of R⁹ and R¹⁰ isnot hydrogen.
 28. The composition of claim 26, wherein R⁷ is optionallysubstituted phenyl and the compound has the following structure (IIa):

wherein R¹¹ and R¹² are the same or different and independentlyhydrogen, hydroxy, carboxy, halo, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl.29.-112. (canceled)
 113. An isolated epithelial cell comprising (i) acalcium-activated chloride channel and (ii) a recombinant cytoplasmicindicator protein that binds halide.
 114. The epithelial cell of claim113 wherein the epithelial cell is an intestinal epithelial cell or apulmonary epithelial cell.
 115. The epithelial cell of claim 113 whereinthe intestinal epithelial cell is an HT-29 cell.
 116. The epithelialcell of claim 113 wherein the cytoplasmic indicator protein is a yellowfluorescent protein (YFP) mutant.
 117. The epithelial cell of claim 116,wherein the YFP mutant is YFP-H148Q/I152L.
 118. The epithelial cell ofclaim 113 wherein the calcium-activated chloride channel is TMEM16A.119. The epithelial cell of claim 118 wherein TMEM16A is human TMEM16A.120. The epithelial cell of claim 113 wherein the recombinantcytoplasmic indicator protein is introduced into the cell by arecombinant expression vector that is a viral vector.
 121. The method ofclaim 120 wherein the viral vector is a retroviral vector.
 122. Themethod of claim 121 wherein the retroviral vector is a lentiviralvector.
 123. A method of identifying an agent that is an inhibitor of acalcium-activated chloride channel comprising: (a) contacting theisolated epithelial cell of claim 113 and a candidate agent in a testsample to permit interaction between the candidate agent and the cell;(b) adding to the test sample (i) at least one calcium-elevating agonistand (ii) iodide, wherein binding of the calcium-elevating agonist to thecell increases the level of calcium ion (Ca²) in the cell; and (c)determining the level of iodide influx in the presence of the candidateagent and comparing the level of iodide influx in the presence of thecandidate agent with the level of iodide influx in the absence of thecandidate agent, wherein a decrease in the level of iodide influx in thepresence of the candidate agent compared with the level of iodide influxin the absence of the candidate agent, indicates that the candidateagent is an inhibitor of the calcium-activated chloride channel. 124.The method of claim 123 wherein the steps of the method are performed ineach of a plurality of reaction vessels in a high throughput screeningarray.
 125. A method of determining influx of an anion in the epithelialcell of claim 113, wherein the anion is halide or NO₃ ⁻, said methodcomprising: (a) contacting the epithelial cell with the anion in thepresence of a calcium-elevating agonist and in the absence of thecalcium-elevating agonist, wherein the cytoplasmic indicator proteinbinds the anion, and wherein binding of the calcium-elevating agonist tothe epithelial cell increase the level of calcium ion (Ca²⁺) in theepithelial cell; and (b) determining the level of anion influx in thepresence of the calcium-elevating agonist and determining the level ofanion influx in the absence of the calcium-elevating agonist, andcomparing the level of anion influx in the presence of thecalcium-elevating agonist to the level of anion influx in the absence ofthe calcium-elevating agonist, thereby determining influx of the anionin the epithelial cell.